Antisense modulation of MARK3 expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of MARK3. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding MARK3. Methods of using these compounds for modulation of MARK3 expression and for treatment of diseases associated with expression of MARK3 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of MARK3. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding MARK3. Such compounds have been shown to modulate the expression of MARK3.

BACKGROUND OF THE INVENTION

[0002] Microtubules are intracellular cytoskeletal components that participate in a variety of cellular processes, such as regulating cell shape, motility, and polarity, serving as tracks for cellular transport, and coordinating the dynamic chromosome movements of mitosis. Central to microtubule function is the property of dynamic instability. Microtubules transition between stable and dynamic states, and these transitions are modulated, in part, by structural microtubule-associated proteins (MAPs). Structural MAPs are filamentous proteins that lack enzymatic activity but bind reversibly to microtubules and stabilize them by promoting tubulin polymerization. The phosphorylation of MAPs influences their microtubule-stabilizing capacity. In mitotic cells, where the microtubule turnover rate increases 18-fold, MAPs exhibit a several-fold higher degree of phosphorylation. Microtubule-affinity-regulating kinases (MARKS) are serine/threonine kinases that phosphorylate the tubulin-binding domain of MAPs, thereby causing their detachment from microtubules and leading to increased microtubule dynamics (Drewes et al., Trends Biochem. Sci., 1998, 23, 307-311).

[0003] The structural MAP family includes the neuronal microtubule associated proteins tau and MAP2, as well as MAP4, which is present in all non-neuronal vertebrate cells. The tau protein is particularly well-studied because phosphorylation of tau at a single residue, Ser-262, dramatically reduced microtubule binding, and phosphorylation of Ser-262 is elevated in tau isolated from the neurofibrillary tangles of Alzheimer's disease. It is believed that the neuronal pathology of this disease may be due to a loss of the ability of tau to bind microtubules. A major kinase activity that phosphorylated the neuronal MAPs tau and MAP2, as well as the ubiquitous MAP4, and which caused rapid detachment of all three MAPs from microtubules and resulted in dynamic instability, was purified, and this lead to the identification of the first MARK protein (Drewes et al., Cell, 1997, 89, 297-308). A family of four related genes, MARK1-4, has since been identified (Drewes et al., Trends Biochem. Sci., 1998, 23, 307-311).

[0004] Because of their involvement in MAP activity and microtubule functions such as cellular morphogenesis and the generation and/or maintenance of cell polarity, the MARK proteins can also serve as markers of differentiating cells (Drewes et al., Trends Biochem. Sci., 1998, 23, 307-311). MARK3 was originally identified as such a marker. In a study of the multiphasic process of human pancreas carcinogenesis, a panel of monoclonal antibodies was developed and used for the detection and characterization of tumorigenic stage. One antibody in this panel had a strong affinity for a membrane-associated protein of 78 kDa which was found to mislocalize in and eventually be lost from tumor cells. Carcinogenesis in fetal pancreas cells is associated with a loss of epithelial polarity, and a loss of this marker correlates with the tumorigenic phenotype (Parsa, Cancer Res., 1988, 48, 2265-2272). This p78 marker present in normal and nontumorigenic cells, but absent from tumorigenic cells and primary carcinomas of human pancreas, was later found to bear homology to the MARK kinases involved in specific phosphorylation of MAPs, and was thus called MARK3 (also known as MAP/microtubule affinity-regulating kinase 3, microtubule-associated protein/microtubule affinity-regulating kinase 3, Cdc25C-associated protein kinase 1, CTAK1, c-TAK1, C-Takl, Cdc twenty-five C associated protein kinase, ELKL motif kinase 2 long form, Emk2, and ETK-1) (Drewes et al., Trends Biochem. Sci., 1998, 23, 307-311; Ono et al., Cytogenet. Cell Genet., 1997, 79, 101-102).

[0005] Independently, the rat MARK3 protein was purified as the Cdc25C-associated protein kinase, CTAK1, suggesting that MARK3 plays a role in cell cycle regulation (Ogg et al., J. Biol. Chem., 1994, 269, 30461-30469). Progression of the eukaryotic cell cycle is controlled by the sequential activation of cyclin-dependent kinases, and human Cdc25C is a dual-specificity protein phosphatase that triggers entry into mitosis by dephosphorylating and thus activating the Cdc2 cyclin-dependent kinase. The Cdc25C phosphatase is itself regulated by phosphorylation, and a Cdc25C-associated protein kinase (MARK3) was isolated from rat liver and found to bind human Cdc25C in vitro and in vivo and to phosphorylate it at Ser-216 (Ogg et al., J. Biol. Chem., 1994, 269, 30461-30469). Purified Cdc25C-associated protein kinase from rat liver was subjected to protein sequencing and two homologous proteins were identified in sequence databases, the human p78 marker protein lost in pancreatic carcinomas, and the murine ELKL motif kinase gene. MARK3 was further characterized to belong to the CaMKII/Snf1/AMPK subfamily of protein kinases which share a conserved N-terminal kinase domain, followed by a divergent C-terminal region of unknown function, and a conserved region of about 40 amino acids at their extreme C-termini which ends in ELKL (glutamate-leucine-lysine-leucine) (Peng et al., Cell Growth Differ., 1998, 9, 197-208).

[0006] Human CTAK1 (MARK3) was subsequently cloned from a B-cell cDNA library by using primers designed against human p78, and it was discovered that the MARK3 gene undergoes alternative splicing. Northern analysis revealed two transcripts of approximately 3.8 and 3.1 kilobases, expressed in all human tissues examined, and a 3.0 kilobase transcript was observed in heart tissue. Using antibodies, endogenous MARK3 protein was found to localize to the cytoplasm but not the nucleus of HeLa cells. (Peng et al., Cell Growth Differ., 1998, 9, 197-208). The MARK3 gene was mapped by fluorescence in situ hybridization to human chromosomal band 14q32.3 (Ono et al., Cytogenet. Cell Genet., 1997, 79, 101-102).

[0007] MARK3 phosphorylates Ser-216 of Cdc25C, and phosphorylation of this residue results in a consensus recognition motif for 14-3-3 proteins. The 14-3-3 proteins are believed to regulate Cdc25C function by binding to Cdc25C and retaining it in the cytoplasmic compartment during interphase. Because MARK3 phosphorylates Ser-216 of Cdc25C and promotes 14-3-3 binding, and Cdc25C remains phosphorylated throughout interphase, Cdc25C/14-3-3 complexes are present throughout interphase. MARK3 is localized to the cytoplasm, and its phosphorylation of Ser-216 of Cdc25C allows 14-3-3 to bind and retain Cdc25C in the cytoplasm, negatively regulating the functional interaction between Cdc25C and the Cdc2/Cyclin B complex during interphase and inhibiting mitotic entry. Thus, one function of the MARK3 kinase in vivo may be to regulate the interactions between important cell cycle regulatory proteins (Dalal et al., Mol. Cell. Biol., 1999, 19, 4465-4479; Peng et al., Cell Growth Differ., 1998, 9, 197-208).

[0008] The 14-3-3 binding site within another protein was also found to be a substrate for phosphorylation by MARK3. Binding of the 14-3-3□ protein to protein-tyrosine phosphatase 1 (PTPHl) is greatly enhanced by pretreating PTPH1 with MARK3. In addition to substantiating the hypothesis that MARK3 phosphorylates and regulates 14-3-3 binding sites, this interaction between the MARK3 kinase and PTPH1 indicates a link between serine/threonine and tyrosine phosphorylation-dependent signaling pathways (Zhang et al., J. Biol. Chem., 1997, 272, 27281-27287).

[0009] Disclosed and claimed in U.S. Pat. No. 5,863,729 is a DNA sequence encoding the amino acid sequence of MARK3, a transformed cell comprising said DNA sequence combined with a heterologous control sequence for expression in said cell, and a method for detecting and quantifying MARK3 expression in a cell or tissue by hybridizing mRNA with MARK3 DNA probes (Piwnica-Worms, 1999).

[0010] Disclosed and claimed in PCT Publication WO 00/73469 is an isolated, enriched, or purified nucleic acid molecule encoding the MARK3 kinase, and a nucleic acid molecule wherein said nucleic acid molecule comprises a nucleotide sequence that is the complement of the MARK3 DNA sequence, and said complementary sequence except that it lacks one or more, but not all, of a domain selected from the group consisting of an N-terminal domain, a catalytic domain, a C-terminal domain, a coiled-coil structure region, a proline-rich region, a spacer region, an insert, and a C-terminal tail. Further claimed is a method for detection of MARK3 in a sample as a diagnostic tool for a disease or disorder, wherein said method comprises contacting said sample with a nucleic acid probe which hybridizes to the MARK3 DNA sequence. Generally disclosed are substances useful for treatment of disorders or diseases that modulate the activity of the polypeptides including antisense oligonucleotides, and transgenic nonhuman mammals containing a antisense nucleic acid transgene for regulating the expression of MARK3 (Plowman et al., 2000).

[0011] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MARK3. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting MARK3 function.

[0012] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of MARK3 expression.

[0013] The present invention provides compositions and methods for modulating MARK3 expression.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding MARK3, and which modulate the expression of MARK3. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of MARK3 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of MARK3 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding MARK3, ultimately modulating the amount of MARK3 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding MARK3. As used herein, the terms “target nucleic acid” and “nucleic acid encoding MARK3” encompass DNA encoding MARK3, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of MARK3. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of gene expression and mRNA is a preferred target.

[0016] It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding MARK3. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding MARK3, regardless of the sequence(s) of such codons.

[0017] It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.

[0018] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.

[0019] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It has also been found that introns can be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.

[0020] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and extronic regions.

[0021] Upon excision of one or more exon or intron regions or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0022] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.

[0023] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0024] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.

[0025] An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. It is preferred that the antisense compounds of the present invention comprise at least 80% sequence complementarity to a target region within the target nucleic acid, moreover that they comprise 90% sequence complementarity and even more comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0026] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The sites to which these preferred antisense compounds are specifically hybridizable are hereinbelow referred to as “preferred target regions” and are therefore preferred sites for targeting. As used herein the term “preferred target region” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target regions represent regions of the target nucleic acid which are accessible for hybridization.

[0027] While the specific sequences of particular preferred target regions are set forth below, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target regions may be identified by one having ordinary skill.

[0028] Target regions 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target regions are considered to be suitable preferred target regions as well.

[0029] Exemplary good preferred target regions include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly good preferred target regions are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred target regions illustrated herein will be able, without undue experimentation, to identify further preferred target regions. In addition, one having ordinary skill in the art will also be able to identify additional compounds, including oligonucleotide probes and primers, that specifically hybridize to these preferred target regions using techniques available to the ordinary practitioner in the art.

[0030] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.

[0031] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0032] Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0033] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0034] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.

[0035] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

[0036] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides from about 8 to about 50 nucleobases, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.

[0037] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0038] Exemplary preferred antisense compounds include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0039] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are herein identified as preferred embodiments of the invention. While specific sequences of the antisense compounds are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred antisense compounds may be identified by one having ordinary skill.

[0040] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. In addition, linear structures may also have internal nucleobase complementarity and may therefore fold in a manner as to produce a double stranded structure. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0041] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0042] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0043] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0044] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0045] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0046] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0047] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0048] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(m)O]CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃) 2 group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0049] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0050] A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0051] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0052] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0053] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0054] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0055] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as interferon-induced RNAseL which cleaves both cellular and viral RNA. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0056] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0057] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0058] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0059] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0060] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0061] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

[0062] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci., 1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.

[0063] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.

[0064] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of MARK3 is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.

[0065] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding MARK3, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding MARK3 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of MARK3 in a sample may also be prepared.

[0066] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.

[0067] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.

[0068] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.

[0069] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0070] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0071] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0072] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0073] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.

[0074] Emulsions

[0075] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 Am in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

[0076] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0077] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

[0078] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0079] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0080] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0081] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0082] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0083] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0084] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0085] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0086] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

[0087] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

[0088] Liposomes

[0089] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

[0090] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[0091] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[0092] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0093] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0094] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

[0095] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.

[0096] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0097] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0098] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0099] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0100] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0101] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0102] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).

[0103] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C₁₂15G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0104] A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.

[0105] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0106] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0107] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0108] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0109] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0110] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0111] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0112] Penetration Enhancers

[0113] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

[0114] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

[0115] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0116] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0117] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0118] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0119] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0120] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.

[0121] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

[0122] Carriers

[0123] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0124] Excipients

[0125] In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0126] Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0127] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

[0128] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0129] Other Components

[0130] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0131] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0132] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0133] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0134] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0135] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0136] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites

[0137] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, optimized synthesis cycles were developed that incorporate multiple steps coupling longer wait times relative to standard synthesis cycles.

[0138] The following abbreviations are used in the text: thin layer chromatography (TLC), melting point (MP), high pressure liquid chromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar), methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethyl formamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF).

[0139] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC) nucleotides were synthesized according to published methods (Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.) or prepared as follows:

[0140] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for 5-methyl dC Amidite

[0141] To a 50 L glass reactor equipped with air stirrer and Ar gas line was added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) at ambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol, 1.05 eq) was added as a solid in four portions over 1 h. After 30 min, TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent and by-products and 2% 3¹,5′-bis DMT product (R_(f) in EtOAc 0.45, 0.05, 0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂ were added with stirring (pH of the aqueous layer 7.5). An additional 18 L of water was added, the mixture was stirred, the phases were separated, and the organic layer was transferred to a second 50 L vessel. The aqueous layer was extracted with additional CH₂Cl₂ (2×2 L). The combined organic layer was washed with water (10 L) and then concentrated in a rotary evaporator to approx. 3.6 kg total weight. This was redissolved in CH₂Cl₂ (3.5 L), added to the reactor followed by water (6 L) and hexanes (13 L). The mixture was vigorously stirred and seeded to give a fine white suspended solid starting at the interface. After stirring for 1 h, the suspension was removed by suction through a ½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cm Coors Buchner funnel, washed with water (2×3 L) and a mixture of hexanes—CH₂Cl₂ (4:1, 2×3 L) and allowed to air dry overnight in pans (1″ deep). This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to a constant weight of 2072 g (93%) of a white solid, (mp 122-124° C.). TLC indicated a trace contamination of the bis DMT product. NMR spectroscopy also indicated that 1-2 mole percent pyridine and about 5 mole percent of hexanes was still present.

[0142] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-methyl-dC Amidite

[0143] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and an Ar gas line was added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrous acetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30 minutes while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition. The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R_(f) 0.43 to 0.84 of starting material and silyl product, respectively). Upon completion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60 min so as to maintain the temperature between −20° C. and −10° C. during the strongly exothermic process, followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h. TLC indicated a complete conversion to the triazole product (R_(f) 0.83 to 0.34 with the product spot glowing in long wavelength UV light). The reaction mixture was a peach-colored thick suspension, which turned darker red upon warming without apparent decomposition. The reaction was cooled to −15° C. internal temperature and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The combined water layers were back-extracted with EtOAc (6 L). The water layer was discarded and the organic layers were concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The second half of the reaction was treated in the same way. Each residue was dissolved in dioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight (although the reaction is complete within 1 h).

[0144] TLC indicated a complete reaction (product R_(f) 0.35 in EtOAc-MeOH 4:1). The reaction solution was concentrated on a rotary evaporator to a dense foam. Each foam was slowly redissolved in warm EtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, and extracted with water (2×4L) to remove the triazole by-product. The water was back-extracted with EtOAc (2 L). The organic layers were combined and concentrated to about 8 kg total weight, cooled to 0° C. and seeded with crystalline product. After 24 hours, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L) until a white powder was left and then washed with ethyl ether (2×3L). The solid was put in pans (1″ deep) and allowed to air dry overnight. The filtrate was concentrated to an oil, then redissolved in EtOAc (2 L), cooled and seeded as before. The second crop was collected and washed as before (with proportional solvents) and the filtrate was first extracted with water (2×1L) and then concentrated to an oil. The residue was dissolved in EtOAc (1 L) and yielded a third crop which was treated as above except that more washing was required to remove a yellow oily layer.

[0145] After air-drying, the three crops were dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g, respectively) and combined to afford 2550 g (85%) of a white crystalline product (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity. The mother liquor still contained mostly product (as determined by TLC) and a small amount of triazole (as determined by NMR spectroscopy), bis DMT product and unidentified minor impurities. If desired, the mother liquor can be purified by silica gel chromatography using a gradient of MeOH (0-25%) in EtOAc to further increase the yield.

[0146] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-methyl dC Amidite

[0147] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000 g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambient temperature in a 50 L glass reactor vessel equipped with an air stirrer and argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86 mol, 1.05 eq) was added and the reaction was stirred at ambient temperature for 8 h. TLC (CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; R_(f) 0.25) indicated approx. 92% complete reaction. An additional amount of benzoic anhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLC indicated approx. 96% reaction completion. The solution was diluted with EtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added with stirring, and the mixture was extracted with water (15 L, then 2×10 L). The aqueous layer was removed (no back-extraction was needed) and the organic layer was concentrated in 2×20 L rotary evaporator flasks until a foam began to form. The residues were coevaporated with acetonitrile (1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a dense foam. High pressure liquid chromatography (HPLC) revealed a contamination of 6.3% of N4, 3′-O-dibenzoyl product, but very little other impurities.

[0148] THe product was purified by Biotage column chromatography (5 kg Biotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product (800 g),dissolved in CH₂Cl₂ (2 L), was applied to the column. The column was washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1 solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractions containing the product were collected, and any fractions containing the product and impurities were retained to be resubjected to column chromatography. The column was re-equilibrated with the original 65:35:1 solvent mixture (17 kg). A second batch of crude product (840 g) was applied to the column as before. The column was washed with the following solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1 (10 kg), and 99:1 EtOAc:TEA (15 kg). The column was reequilibrated as above, and a third batch of the crude product (850 g) plus impure fractions recycled from the two previous columns (28 g) was purified following the procedure for the second batch. The fractions containing pure product combined and concentrated on a 20L rotary evaporator, co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25° C.) to a constant weight of 2023 g (85%) of white foam and 20 g of slightly contaminated product from the third run. HPLC indicated a purity of 99.8% with the balance as the diBenzoyl product.

[0149] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC Amidite)

[0150] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4-benzoyl-5-methylcytidine (998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (300 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (15 ml) was added and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2.5 L) and water (600 ml), and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (7.5 L) and hexane (6 L). The two layers were separated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L) and water (3×2 L), and the phases were separated. The organic layer was dried (Na₂SO₄), filtered and rotary evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried to a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g an off-white foam solid (96%).

[0151] 2′-Fluoro Amidites

[0152] 2′-Fluorodeoxyadenosine Amidites

[0153] 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. The preparation of 2′-fluoropyrimidines containing a 5-methyl substitution are described in U.S. Pat. No. 5,861,493. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and whereby the 2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a 2′-beta-triflate group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0154] 2′-Fluorodeoxyguanosine

[0155] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate isobutyrylarabinofuranosylguanosine. Alternatively, isobutyrylarabinofuranosylguanosine was prepared as described by Ross et al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give isobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.

[0156] 2′-Fluorouridine

[0157] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0158] 2′-Fluorodeoxycytidine

[0159] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0160] 2′-O-(2-Methoxyethyl) Modified Amidites

[0161] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwise known as MOE amidites) are prepared as follows, or alternatively, as per the methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

[0162] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine Intermediate

[0163] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol), tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60 g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12 L three necked flask and heated to 130° C. (internal temp) at atmospheric pressure, under an argon atmosphere with stirring for 21 h. TLC indicated a complete reaction. The solvent was removed under reduced pressure until a sticky gum formed (50-85° C. bath temp and 100-11 mm Hg) and the residue was redissolved in water (3 L) and heated to boiling for 30 min in order the hydrolyze the borate esters. The water was removed under reduced pressure until a foam began to form and then the process was repeated. HPLC indicated about 77% product, 15% dimer (5′ of product attached to 2′ of starting material) and unknown derivatives, and the balance was a single unresolved early eluting peak.

[0164] The gum was redissolved in brine (3 L), and the flask was rinsed with additional brine (3 L). The combined aqueous solutions were extracted with chloroform (20 L) in a heavier-than continuous extractor for 70 h. The chloroform layer was concentrated by rotary evaporation in a 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH (400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolved at which point the vacuum was lowered to about 0.5 atm. After 2.5 L of distillate was collected a precipitate began to form and the flask was removed from the rotary evaporator and stirred until the suspension reached ambient temperature. EtOAc (2 L) was added and the slurry was filtered on a 25 cm table top Buchner funnel and the product was washed with EtOAc (3×2 L). The bright white solid was air dried in pans for 24 h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to afford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

[0165] The brine layer in the 20 L continuous extractor was further extracted for 72 h with recycled chloroform. The chloroform was concentrated to 120 g of oil and this was combined with the mother liquor from the above filtration (225 g), dissolved in brine (250 mL) and extracted once with chloroform (250 mL). The brine solution was continuously extracted and the product was crystallized as described above to afford an additional 178 g of crystalline product containing about 2% of thymine. The combined yield was 1827 g (69.4%). HPLC indicated about 99.5% purity with the balance being the dimer.

[0166] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate

[0167] In a 50 L glass-lined steel reactor, 2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol), lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile (15 L). The solution was stirred rapidly and chilled to −10° C. (internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g, 5.21 mol) was added as a solid in one portion. The reaction was allowed to warm to −2° C. over 1 h. (Note: The reaction was monitored closely by TLC (EtOAc) to determine when to stop the reaction so as to not generate the undesired bis-DMT substituted side product). The reaction was allowed to warm from −2 to 3° C. over 25 min. then quenched by adding MeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L). The solution was transferred to a clear 50 L vessel with a bottom outlet, vigorously stirred for 1 minute, and the layers separated. The aqueous layer was removed and the organic layer was washed successively with 10% aqueous citric acid (8 L) and water (12 L). The product was then extracted into the aqueous phase by washing the toluene solution with aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueous layer was overlayed with toluene (12 L) and solid citric acid (8 moles, 1270 g) was added with vigorous stirring to lower the pH of the aqueous layer to 5.5 and extract the product into the toluene. The organic layer was washed with water (10 L) and TLC of the organic layer indicated a trace of DMT-O-Me, bis DMT and dimer DMT.

[0168] The toluene solution was applied to a silica gel column (6 L sintered glass funnel containing approx. 2 kg of silica gel slurried with toluene (2 L) and TEA(25 mL)) and the fractions were eluted with toluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flask placed below the column. The first EtOAc fraction containing both the desired product and impurities were resubjected to column chromatography as above. The clean fractions were combined, rotary evaporated to a foam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven (0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMR spectroscopy indicated a 0.25 mole % remainder of acetonitrile (calculates to be approx. 47 g) to give a true dry weight of 2803 g (96%). HPLC indicated that the product was 99.41% pure, with the remainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and no detectable dimer DMT or 3′-O-DMT.

[0169] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T Amidite)

[0170] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine (1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solution was co-evaporated with toluene (200 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g, 1.0 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (20 ml) was added and the solution was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (3.5 L) and water (600 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.6 L) and extracted with the mixture of toluene (12 L) and hexanes (9 L). The upper layer was washed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organic layer was dried (Na₂SO₄), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of an off-white foamy solid (95%).

[0171] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate

[0172] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and argon gas line was added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine (2.616 kg, 4.23 mol, purified by base extraction only and no scrub column), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7 mol, 3.0 eq) was added over 30 min. while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). (Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition). The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc, R_(f) 0.68 and 0.87 for starting material and silyl product, respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was added slowly over 60 min so as to maintain the temperature between −20° C. and −10° C. (note: strongly exothermic), followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h, at which point it was an off-white thick suspension. TLC indicated a complete conversion to the triazole product (EtOAc, R_(f) 0.87 to 0.75 with the product spot glowing in long wavelength UV light). The reaction was cooled to −15° C. and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The second half of the reaction was treated in the same way. The combined aqueous layers were back-extracted with EtOAc (8 L) The organic layers were combined and concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The residue was dissolved in dioxane (2 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight

[0173] TLC indicated a complete reaction (CH₂Cl₂-acetone-MeOH, 20:5:3, R_(f) 0.51). The reaction solution was concentrated on a rotary evaporator to a dense foam and slowly redissolved in warm CH₂Cl₂ (4 L, 40° C.) and transferred to a 20 L glass extraction vessel equipped with a air-powered stirrer. The organic layer was extracted with water (2×6 L) to remove the triazole by-product. (Note: In the first extraction an emulsion formed which took about 2 h to resolve). The water layer was back-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water (3 L). The combined organic layer was concentrated in 2×20 L flasks to a gum and then recrystallized from EtOAc seeded with crystalline product. After sitting overnight, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc until a white free-flowing powder was left (about 3×3 L). The filtrate was concentrated to an oil recrystallized from EtOAc, and collected as above. The solid was air-dried in pans for 48 h, then further dried in a vacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a bright white, dense solid (86%). An HPLC analysis indicated both crops to be 99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAc remained.

[0174] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-4-benzoyl-5-methyl-cytidine Penultimate Intermediate:

[0175] Crystalline 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g, 1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperature and stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94 mol) was added in one portion. The solution clarified after 5 hours and was stirred for 16 h. HPLC indicated 0.45% starting material remained (as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoic anhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicated no starting material was present. TEA (450 mL, 3.24 mol) and toluene (6 L) were added with stirring for 1 minute. The solution was washed with water (4×4 L), and brine (2×4 L). The organic layer was partially evaporated on a 20 L rotary evaporator to remove 4 L of toluene and traces of water. HPLC indicated that the bis benzoyl side product was present as a 6% impurity. The residue was diluted with toluene (7 L) and anhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g, 1.75 mol) was added in one portion with stirring at ambient temperature over 1 h. The reaction was quenched by slowly adding then washing with aqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed by aqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). The organic layer was concentrated on a 20 L rotary evaporator to about 2 L total volume. The residue was purified by silica gel column chromatography (6 L Buchner funnel containing 1.5 kg of silica gel wetted with a solution of EtOAc-hexanes-TEA(70:29:1)). The product was eluted with the same solvent (30 L) followed by straight EtOAc (6 L). The fractions containing the product were combined, concentrated on a rotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2 mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLC indicated a purity of >99.7%.

[0176] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C Amidite)

[0177] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine (1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporated with toluene (300 ml) at 50° C. under reduced pressure. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3 L>. The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40 v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1336 g of an off-white foam (97%).

[0178] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A Amdite)

[0179] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosine (purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5 mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene (300 ml) at 50° C. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (1.4 L) and extracted with the mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered and evaporated to a sticky foam. The residue was co-evaporated with acetonitrile (2.5 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of an off-white foam solid (96%).

[0180] Prepartion of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite)

[0181] 5′-o-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrlguanosine (purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (200 ml) at 50° C., cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2 L) and water (600 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (2 L) and extracted with a mixture of toluene (10 L) and hexanes (5 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and the solution was washed with water (3×4 L). The organic layer was dried (Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) was added, the mixture was shaken for 10 min, and the supernatant liquid was decanted. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1660 g of an off-white foamy solid (91%).

[0182] 2′-O-(Aminooxyethyl) Nucleoside Amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0183] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites

[0184] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.

[0185] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0186] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (R_(f) 0.22, EtOAc) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between CH₂Cl₂ (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether (600 mL) and cooling the solution to −10° C. afforded a white crystalline solid which was collected by filtration, washed with ethyl ether (3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g of white solid (74.8%). TLC and NMR spectroscopy were consistent with pure product.

[0187] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0188] In the fume hood, ethylene glycol (350 mL, excess) was added cautiously with manual stirring to a 2 L stainless steel pressure reactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). (Caution: evolves hydrogen gas). 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure <100 psig). The reaction vessel was cooled to ambient temperature and opened. TLC (EtOAc, R_(f) 0.67 for desired product and R_(f) 0.82 for ara-T side product) indicated about 70% conversion to the product. The solution was concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. (Alternatively, once the THF has evaporated the solution can be diluted with water and the product extracted into EtOAc). The residue was purified by column chromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, evaporated and dried to afford 84 g of a white crisp foam (50%), contaminated starting material (17.4 g, 12% recovery) and pure reusable starting material (20 g, 13% recovery). TLC and NMR spectroscopy were consistent with 99% pure product.

[0189] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0190] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P₂O₅ under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle). Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture with the rate of addition maintained such that the resulting deep red coloration is just discharged before adding the next drop. The reaction mixture was stirred for 4 hrs., after which time TLC (EtOAc:hexane, 60:40) indicated that the reaction was complete. The solvent was evaporated in vacuuo and the residue purified by flash column chromatography (eluted with 60:40 EtOAc:hexane), to yield 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%) upon rotary evaporation.

[0191] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0192] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate washed with ice cold CH₂Cl₂, and the combined organic phase was washed with water and brine and dried (anhydrous Na₂SO₄). The solution was filtered and evaporated to afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. The solvent was removed under vacuum and the residue was purified by column chromatography to yield 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotary evaporation.

[0193] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine

[0194] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C. under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and the reaction mixture was stirred. After 10 minutes the reaction was warmed to room temperature and stirred for 2 h. while the progress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product was extracted with EtOAc (2×20 mL). The organic phase was dried over anhydrous Na₂SO₄, filtered, and evaporated to dryness. This entire procedure was repeated with the resulting residue, with the exception that formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolution of the residue in the PPTS/MeOH solution. After the extraction and evaporation, the residue was purified by flash column chromatography and (eluted with 5% MeOH in CH₂Cl₂) to afford 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%) upon rotary evaporation.

[0195] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0196] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24 hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removed under vacuum and the residue purified by flash column chromatography (eluted with 10% MeOH in CH₂Cl₂) to afford 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotary evaporation of the solvent.

[0197] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0198] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P₂O₅ under high vacuum overnight at 40° C., co-evaporated with anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to the pyridine solution and the reaction mixture was stirred at room temperature until all of the starting material had reacted. Pyridine was removed under vacuum and the residue was purified by column chromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops of pyridine) to yield 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%) upon rotary evaporation.

[0199] 5′-O-DMT-2¹-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0200] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried over P₂O₅ under high vacuum overnight at 40° C. This was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N¹N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 h under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, then the residue was dissolved in EtOAc (70 mL) and washed with 5% aqueous NaHCO₃ (40 mL). The EtOAc layer was dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue obtained was purified by column chromatography (EtOAc as eluent) to afford 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%) upon rotary evaporation.

[0201] 2′-(Aminooxyethoxy) Nucleoside Amidites

[0202] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.

[0203] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0204] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with aminor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 Al 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may be phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0205] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites

[0206] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.

[0207] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine

[0208] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowly added to a solution of borane in tetrahydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves as the solid dissolves). O₂-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) were added and the bomb was sealed, placed in an oil bath and heated to 155° C. for 26 h. then cooled to room temperature. The crude solution was concentrated, the residue was diluted with water (200 mL) and extracted with hexanes (200 mL). The product was extracted from the aqueous layer with EtOAc (3×200 mL) and the combined organic layers were washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (eluted with 5:100:2 MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combined and evaporated to afford the product as a white solid.

[0209] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl Uridine

[0210] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. The reaction mixture was poured into water (200 mL) and extracted with CH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers were washed with saturated NaHCO₃ solution, followed by saturated NaCl solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography (eluted with 5:100:1 MeOH/CH₂Cl₂/TEA) to afford the product.

[0211] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0212] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere of argon. The reaction mixture was stirred overnight and the solvent evaporated. The resulting residue was purified by silica gel column chromatography with EtOAc as the eluent to afford the title compound.

Example 2

[0213] Oligonucleotide Synthesis

[0214] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0215] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄oAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0216] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0217] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0218] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0219] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference. 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0220] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0221] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

[0222] Oligonucleoside Synthesis

[0223] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0224] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0225] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0226] PNA Synthesis

[0227] Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.

Example 5

[0228] Synthesis of Chimeric Oligonucleotides

[0229] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0230] [2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0231] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0232] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0233] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0234] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0235] [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0236] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0237] oligonucleotide Isolation

[0238] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0239] Oligonucleotide Synthesis—96 Well Plate Format

[0240] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0241] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0242] Oligonucleotide Analysis—96-Well Plate Format

[0243] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0244] Cell Culture and Oligonucleotide Treatment

[0245] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0246] T-24 Cells:

[0247] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0248] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0249] A549 Cells:

[0250] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0251] NHDF Cells:

[0252] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0253] HEK Cells:

[0254] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0255] Treatment with Antisense Compounds:

[0256] When cells reached 70% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0257] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

[0258] Analysis of Oligonucleotide Inhibition of MARK3 Expression

[0259] Antisense modulation of MARK3 expression can be assayed in a variety of ways known in the art. For example, MARK3 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0260] Protein levels of MARK3 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to MARK3 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons, Inc., 1997). Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997).

[0261] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998). Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991).

Example 11

[0262] 15 Poly(A)+ mRNA Isolation

[0263] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0264] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

Example 12

[0265] Total RNA Isolation

[0266] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RWl was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 170 μL water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0267] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0268] Real-Time Quantitative PCR Analysis of MARK3 mRNA Levels

[0269] Quantitation of MARK3 mRNA levels was determined by real-time quantitative PCR using the ABI PRISMM 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0270] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0271] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer (—MgCl2), 6.6 mM MgCl2, 375 μM each of DATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0272] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreenTM RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreenTM are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0273] In this assay, 170 μL of RiboGreenTM working reagent (RiboGreenTM reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.

[0274] Probes and primers to human MARK3 were designed to hybridize to a human MARK3 sequence, using published sequence information (GenBank accession number U64205.1, incorporated herein as SEQ ID NO:4). For human MARK3 the PCR primers were:

[0275] forward primer: TGACCATGCTGGACCAGCTA (SEQ ID NO: 5)

[0276] reverse primer: TCACCATCTGCAGTGCTTGTCT (SEQ ID NO: 6) and the

[0277] PCR probe was: FAM-CCTTCTGTTGTGGCGTATCCGAAAAGGA-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were:

[0278] forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8)

[0279] reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the

[0280] PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC— TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0281] Northern Blot Analysis of MARK3 mRNA Levels

[0282] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0283] To detect human MARK3, a human MARK3 specific probe was prepared by PCR using the forward primer TGACCATGCTGGACCAGCTA (SEQ ID NO: 5) and the reverse primer TCACCATCTGCAGTGCTTGTCT (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0284] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0285] Antisense Inhibition of Human MARK3 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0286] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human MARK3 RNA, using published sequences (GenBank accession number U64205.1, incorporated herein as SEQ ID NO: 4, GenBank accession number NM_(—)002376.1, incorporated herein as SEQ ID NO: 11, GenBank accession number AF159295.1, incorporated herein as SEQ ID NO: 12, GenBank accession number BF083244.1, incorporated herein as SEQ ID NO: 13, GenBank accession number AF170723.1, incorporated herein as SEQ ID NO: 14, and a genomic sequence representing nucleotides 177000-296500 of GenBank accession number NT_(—)028360.1, the complement of which is incorporated herein as SEQ ID NO: 15). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human MARK3 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which T-24 cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human MARK3 mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQ ID TARGET % SEQ SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB ID NO NO 151465 Coding 4 398 gtctcgttcattcaccgttg 11 16 2 151466 Coding 4 1814 gcgttcaggaatatccgcct 19 17 2 151467 Coding 4 2040 gactggcagtgcctcttggg 71 18 2 151468 Coding 4 2168 gagattagtggagcctcggc 89 19 2 151469 Coding 4 896 cttgaggtctcgatgtacga 77 20 2 151470 Coding 4 2155 cctcggcttcgagtctggga 95 21 2 151471 Coding 4 498 ctgcacaggaggctatagag 68 22 2 151472 Coding 4 1792 ttaggattacttgcattccc 98 23 2 151473 Coding 4 1665 gactccttttcggatacgcc 79 24 2 151474 5′UTR 4 345 ttaattctgcacaatgcgag 91 25 2 151475 Coding 4 590 tgtaaggatatgtcttgcca 35 26 2 151476 5′UTR 4 341 ttctgcacaatgcgagggtc 75 27 2 151477 Coding 4 1977 ttgaagcaactggagttctc 22 28 2 151478 Coding 4 500 atctgcacaggaggctatag 56 29 2 151479 3′UTR 4 2590 gaaaacactttacttgctac 25 30 2 151480 Coding 4 2134 aatggtgtggcttcatggga 75 31 2 151481 Coding 4 988 gtgtcgagtttaccgccaac 83 32 2 151482 Coding 4 1868 tcgtgtcattccaccagatg 91 33 2 151483 Coding 4 1762 ctggctggagcaattccctt 50 34 2 151484 Coding 4 1345 ttttggtctgagatgtctag 74 35 2 151485 Coding 4 1899 tagttctctcactgcaaaca 51 36 2 151486 Coding 4 2224 ttttgctcagcagatacatt 28 37 2 151487 Coding 4 495 cacaggaggctatagagttt 82 38 2 151488 5′UTR 4 218 ccgttctagatcccgggcct 62 39 2 151489 Coding 4 2050 aaagtgctacgactggcagt 12 40 2 151490 Coding 4 795 gtgcaaccaaatagtcaaat 65 41 2 151491 3′UTR 4 2600 cagtgttcaggaaaacactt 12 42 2 151492 Coding 4 550 cccttgccgattgttttcaa 68 43 2 151493 Coding 4 1296 cttcttcatgccctgcattg 83 44 2 151494 Coding 4 469 cgagctcctgagcggctggt 91 45 2 151495 Coding 4 1233 ttggatttagcaccaggaaa 70 46 2 151496 Coding 4 1998 ctgcactactgatactgtgt 87 47 2 151497 Coding 4 1877 agtatttcgtcgtgtcattc 69 48 2 151498 Coding 4 1090 gtgtataaaatgacccccag 79 49 2 151499 Coding 4 1829 agtggagcttttcttgcgtt 70 50 2 151500 Coding 4 2208 cattgcgactccttgtgagt 59 51 2 151501 Coding 4 647 tagacttgttggattcaact 5 52 2 199661 Start 4 367 ctagtggacattttactgca 72 53 2 Codon 199662 Coding 4 609 ttattgcaacctctctgcct 36 54 2 199663 Coding 4 663 ctctgaagagcttttgtaga 50 55 2 199664 Coding 4 1476 tagcatccagctctgaagat 0 56 2 199665 Coding 4 1848 ctgtgttactactagggaca 67 57 2 199666 Stop 4 2555 atcactgggttacagcttta 63 58 2 Codon 199667 Coding 11 1272 tcggcctaacctctgaagat 0 59 2 199668 3′UTR 11 2488 ggttgcacatctttaatgta 10 60 2 199669 3′UTR 11 2647 ggtactagtaatgactggct 23 61 2 199670 3′UTR 11 2663 gatgatctcccgcagaggta 10 62 2 199671 5′UTR 12 363 gatgcccttagatgtccggg 0 63 2 199672 5′UTR 12 393 ccacccgagattgagcaata 0 64 2 199673 5′UTR 12 562 gatagctctttctcgattcc 9 65 2 199674 5′UTR 12 719 ggaaaccaaagtctttgggt 0 66 2 199675 Coding 12 1977 cttgacaaccctgtctaaat 0 67 2 199676 Coding 12 2049 ctgcagacacaataaatgta 0 68 2 199677 intron 13 12 gtttagttaaccaaacacga 0 69 2 199678 intron 13 21 cacctttaggtttagttaac 0 70 2 199679 intron: 13 67 ctctcagttcctttggagag 35 71 2 exon junction 199680 exon: 13 187 acatgattacctctagagtg 46 72 2 intron junction 199681 intron 13 263 ccagtatggcatacaaatca 55 73 2 199682 intron 13 385 tctgataaccgtaatattta 0 74 2 199683 exon: 14 890 tgacatgtttctccttgtga 22 75 2 exon junction 199684 exon: 14 917 agttggaagccttttgataa 0 76 2 exon junction 199685 exon: 14 926 ctcatattcagttggaagcc 67 77 2 exon junction 199686 exon: 14 957 tgcgacttgagccctcatat 37 78 2 exon junction 199687 exon: 14 962 tacattgcgacttgagccct 20 79 2 exon junction 199688 intron 15 25265 attgtgttacagcagcaaaa 62 80 2 199689 intron 15 61215 gacacatttttgtgcacctg 72 81 2 199690 intron: 15 72274 aatacttcacctataggtga 4 82 2 exon junction 199691 intron 15 74052 gagaagttaaatgatagcca 21 83 2 199692 intron: 15 77537 cagacacaatctgaatagga 7 84 2 exon junction 199693 intron: 15 83213 tcggcctaaccttagcaagt 71 85 2 exon junction 199694 intron 15 101800 atcacaatggtctacatata 41 86 2 199695 intron: 15 106909 aggaatagtgctatgagatc 21 87 2 exon junction

[0287] As shown in Table 1, SEQ ID NOs 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 31, 32, 33, 35, 38, 39, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 53, 57, 58, 73, 77, 80, 81 and 85 demonstrated at least 55% inhibition of human MARK3 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “preferred target regions” and are therefore preferred sites for targeting by compounds of the present invention. These preferred target regions are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number of the corresponding target nucleic acid. Also shown in Table 2 is the species in which each of the preferred target regions was found. TABLE 2 Sequence and position of preferred target regions identified in MARK3. TARGET REV SITE SEQ ID TARGET COMP OF SEQ ID ID NO SITE SEQUENCE SEQ ID ACTIVE IN NO 66987 4 2040 cccaagaggcactgccagtc 18 H. sapiens 88 66988 4 2168 gccgaggctccactaatctc 19 H. sapiens 89 66989 4 896 tcgtacatcgagacctcaag 20 H. sapiens 90 66990 4 2155 tcccagactcgaagccgagg 21 H. sapiens 91 66991 4 498 ctctatagcctcctgtgcag 22 H. sapiens 92 66992 4 1792 gggaatgcaagtaatcctaa 23 H. sapiens 93 66993 4 1665 ggcgtatccgaaaaggagtc 24 H. sapiens 94 66994 4 345 ctcgcattgtgcagaattaa 25 H. sapiens 95 66996 4 341 gaccctcgcattgtgcagaa 27 H. sapiens 96 66998 4 500 ctatagcctcctgtgcagat 29 H. sapiens 97 67000 4 2134 tcccatgaagccacaccatt 31 H. sapiens 98 67001 4 988 gttggcggtaaactcgacac 32 H. sapiens 99 67002 4 1868 catctggtggaatgacacga 33 H. sapiens 100 67004 4 1345 ctagacatctcagaccaaaa 35 H. sapiens 101 67007 4 495 aaactctatagcctcctgtg 38 H. sapiens 102 67008 4 218 aggcccgggatctagaacgg 39 H. sapiens 103 67010 4 795 atttgactatttggttgcac 41 H. sapiens 104 67012 4 550 ttgaaaacaatcggcaaggg 43 H. sapiens 105 67013 4 1296 caatgcagggcatgaagaag 44 H. sapiens 106 67014 4 469 accagccgctcaggagctcg 45 H. sapiens 107 67015 4 1233 tttcctggtgctaaatccaa 46 H. sapiens 108 67016 4 1998 acacagtatcagtagtgcag 47 H. sapiens 109 67017 4 1877 gaatgacacgacgaaatact 48 H. sapiens 110 67018 4 1090 ctgggggtcattttatacac 49 H. sapiens 111 67019 4 1829 aacgcaagaaaagctccact 50 H. sapiens 112 67020 4 2208 actcacaaggagtcgcaatg 51 H. sapiens 113 117395 4 367 tgcagtaaaatgtccactag 53 H. sapiens 114 117399 4 1848 tgtccctagtagtaacacag 57 H. sapiens 115 117400 4 2555 taaagctgtaacccagtgat 58 H. sapiens 116 117415 13 263 tgatttgtatgccatactgg 73 H. sapiens 117 117419 14 926 ggcttccaactgaatatgag 77 H. sapiens 118 117422 15 25265 ttttgctgctgtaacacaat 80 H. sapiens 119 117423 15 61215 caggtgcacaaaaatgtgtc 81 H. sapiens 120 117427 15 83213 acttgctaaggttaggccga 85 H. sapiens 121

[0288] As these “preferred target regions” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these sites and consequently inhibit the expression of MARK3.

[0289] In one embodiment, the “preferred target region” may be employed in screening candidate antisense compounds. “Candidate antisense compounds” are those that inhibit the expression of a nucleic acid molecule encoding MARK3 and which comprise at least an 8-nucleobase portion which is complementary to a preferred target region. The method comprises the steps of contacting a preferred target region of a nucleic acid molecule encoding MARK3 with one or more candidate antisense compounds, and selecting for one or more candidate antisense compounds which inhibit the expression of a nucleic acid molecule encoding MARK3. Once it is shown that the candidate antisense compound or compounds are capable of inhibiting the expression of a nucleic acid molecule encoding MARK3, the candidate antisense compound may be employed as an antisense compound in accordance with the present invention.

[0290] According to the present invention, antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.

Example 16

[0291] Western Blot Analysis of MARK3 Protein Levels

[0292] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to MARK3 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

1 121 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial Sequence Antisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 2698 DNA H. sapiens CDS (376)...(2565) 4 gagctgaaat tcgcggtgcg acgggaggga gtggagaagg aggtgagggg gcccaggatc 60 gcggggcgcc ctgaggcaag gggacgccgg tgggtcgaag cgcagcccgc cgcccgcagg 120 ctcggctccg ccactgccgc cctcccggtc tcctcgcctc gggcgccgag gcagggagag 180 aatgagcccc gggacccgcc gggggacggc ccgggccagg cccgggatct agaacggccg 240 tagggggaag ggagccgccc tccccacggc gccttttcgg aactgccgtg gactcgagga 300 cgctggtcgc cggcctccta gggctgtgct gttttgtttt gaccctcgca ttgtgcagaa 360 ttaaagtgca gtaaa atg tcc act agg acc cca ttg cca acg gtg aat gaa 411 Met Ser Thr Arg Thr Pro Leu Pro Thr Val Asn Glu 1 5 10 cga gac act gaa aac cac acg tca cat gga gat ggg cgt caa gaa gtt 459 Arg Asp Thr Glu Asn His Thr Ser His Gly Asp Gly Arg Gln Glu Val 15 20 25 acc tct cgt acc agc cgc tca gga gct cgg tgt aga aac tct ata gcc 507 Thr Ser Arg Thr Ser Arg Ser Gly Ala Arg Cys Arg Asn Ser Ile Ala 30 35 40 tcc tgt gca gat gaa caa cct cac atc gga aac tac aga ctg ttg aaa 555 Ser Cys Ala Asp Glu Gln Pro His Ile Gly Asn Tyr Arg Leu Leu Lys 45 50 55 60 aca atc ggc aag ggg aat ttt gca aaa gta aaa ttg gca aga cat atc 603 Thr Ile Gly Lys Gly Asn Phe Ala Lys Val Lys Leu Ala Arg His Ile 65 70 75 ctt aca ggc aga gag gtt gca ata aaa ata att gac aaa act cag ttg 651 Leu Thr Gly Arg Glu Val Ala Ile Lys Ile Ile Asp Lys Thr Gln Leu 80 85 90 aat cca aca agt cta caa aag ctc ttc aga gaa gta aga ata atg aag 699 Asn Pro Thr Ser Leu Gln Lys Leu Phe Arg Glu Val Arg Ile Met Lys 95 100 105 att tta aat cat ccc aat ata gtg aag tta ttc gaa gtc att gaa act 747 Ile Leu Asn His Pro Asn Ile Val Lys Leu Phe Glu Val Ile Glu Thr 110 115 120 gaa aaa aca ctc tac cta atc atg gaa tat gca agt gga ggt gaa gta 795 Glu Lys Thr Leu Tyr Leu Ile Met Glu Tyr Ala Ser Gly Gly Glu Val 125 130 135 140 ttt gac tat ttg gtt gca cat ggc agg atg aag gaa aaa gaa gca aga 843 Phe Asp Tyr Leu Val Ala His Gly Arg Met Lys Glu Lys Glu Ala Arg 145 150 155 tct aaa ttt aga cag att gtg tct gca gtt caa tac tgc cat cag aaa 891 Ser Lys Phe Arg Gln Ile Val Ser Ala Val Gln Tyr Cys His Gln Lys 160 165 170 cgg atc gta cat cga gac ctc aag gct gaa aat cta ttg tta gat gcc 939 Arg Ile Val His Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala 175 180 185 gat atg aac att aaa ata gca gat ttc ggt ttt agc aat gaa ttt act 987 Asp Met Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser Asn Glu Phe Thr 190 195 200 gtt ggc ggt aaa ctc gac acg ttt tgt ggc agt cct cca tac gca gca 1035 Val Gly Gly Lys Leu Asp Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala 205 210 215 220 cct gag ctc ttc cag ggc aag aaa tat gac ggg cca gaa gtg gat gtg 1083 Pro Glu Leu Phe Gln Gly Lys Lys Tyr Asp Gly Pro Glu Val Asp Val 225 230 235 tgg agt ctg ggg gtc att tta tac aca cta gtc agt ggc tca ctt ccc 1131 Trp Ser Leu Gly Val Ile Leu Tyr Thr Leu Val Ser Gly Ser Leu Pro 240 245 250 ttt gat ggg caa aac cta aag gaa ctg aga gag aga gta tta aga ggg 1179 Phe Asp Gly Gln Asn Leu Lys Glu Leu Arg Glu Arg Val Leu Arg Gly 255 260 265 aaa tac aga att ccc ttc tac atg tct aca gac tgt gaa aac ctt ctc 1227 Lys Tyr Arg Ile Pro Phe Tyr Met Ser Thr Asp Cys Glu Asn Leu Leu 270 275 280 aaa cgt ttc ctg gtg cta aat cca att aaa cgc ggc act cta gag caa 1275 Lys Arg Phe Leu Val Leu Asn Pro Ile Lys Arg Gly Thr Leu Glu Gln 285 290 295 300 atc atg aag gac agg tgg atc aat gca ggg cat gaa gaa gat gaa ctc 1323 Ile Met Lys Asp Arg Trp Ile Asn Ala Gly His Glu Glu Asp Glu Leu 305 310 315 aaa cca ttt gtt gaa cca gag cta gac atc tca gac caa aaa aga ata 1371 Lys Pro Phe Val Glu Pro Glu Leu Asp Ile Ser Asp Gln Lys Arg Ile 320 325 330 gat att atg gtg gga atg gga tat tca caa gaa gaa att caa gaa tct 1419 Asp Ile Met Val Gly Met Gly Tyr Ser Gln Glu Glu Ile Gln Glu Ser 335 340 345 ctt agt aag atg aaa tac gat gaa atc aca gct aca tat ttg tta ttg 1467 Leu Ser Lys Met Lys Tyr Asp Glu Ile Thr Ala Thr Tyr Leu Leu Leu 350 355 360 ggg aga aaa tct tca gag ctg gat gct agt gat tcc agt tct agc agc 1515 Gly Arg Lys Ser Ser Glu Leu Asp Ala Ser Asp Ser Ser Ser Ser Ser 365 370 375 380 aat ctt tca ctt gct aag gtt agg ccg agc agt gat ctc aac aac agt 1563 Asn Leu Ser Leu Ala Lys Val Arg Pro Ser Ser Asp Leu Asn Asn Ser 385 390 395 act ggc cag tct cct cac cac aaa gtg cag aga agt gtt tct tca agc 1611 Thr Gly Gln Ser Pro His His Lys Val Gln Arg Ser Val Ser Ser Ser 400 405 410 caa aag caa aga cgc tac agt gac cat gct gga cca gct att cct tct 1659 Gln Lys Gln Arg Arg Tyr Ser Asp His Ala Gly Pro Ala Ile Pro Ser 415 420 425 gtt gtg gcg tat ccg aaa agg agt cag aca agc act gca gat ggt gac 1707 Val Val Ala Tyr Pro Lys Arg Ser Gln Thr Ser Thr Ala Asp Gly Asp 430 435 440 ctc aaa gaa gat gga att tcc tcc cgg aaa tca agt ggc agt gct gtt 1755 Leu Lys Glu Asp Gly Ile Ser Ser Arg Lys Ser Ser Gly Ser Ala Val 445 450 455 460 gga gga aag gga att gct cca gcc agt ccc atg ctt ggg aat gca agt 1803 Gly Gly Lys Gly Ile Ala Pro Ala Ser Pro Met Leu Gly Asn Ala Ser 465 470 475 aat cct aat aag gcg gat att cct gaa cgc aag aaa agc tcc act gtc 1851 Asn Pro Asn Lys Ala Asp Ile Pro Glu Arg Lys Lys Ser Ser Thr Val 480 485 490 cct agt agt aac aca gca tct ggt gga atg aca cga cga aat act tat 1899 Pro Ser Ser Asn Thr Ala Ser Gly Gly Met Thr Arg Arg Asn Thr Tyr 495 500 505 gtt tgc agt gag aga act aca gct gat aga cac tca gtg att cag aat 1947 Val Cys Ser Glu Arg Thr Thr Ala Asp Arg His Ser Val Ile Gln Asn 510 515 520 ggc aaa gaa aac agc act att cct gat cag aga act cca gtt gct tca 1995 Gly Lys Glu Asn Ser Thr Ile Pro Asp Gln Arg Thr Pro Val Ala Ser 525 530 535 540 aca cac agt atc agt agt gca gcc acc cca gat cga atc cgc ttc cca 2043 Thr His Ser Ile Ser Ser Ala Ala Thr Pro Asp Arg Ile Arg Phe Pro 545 550 555 aga ggc act gcc agt cgt agc act ttc cac ggc cag ccc cgg gaa cgg 2091 Arg Gly Thr Ala Ser Arg Ser Thr Phe His Gly Gln Pro Arg Glu Arg 560 565 570 cga acc gca aca tat aat ggc cct cct gcc tct ccc agc ctg tcc cat 2139 Arg Thr Ala Thr Tyr Asn Gly Pro Pro Ala Ser Pro Ser Leu Ser His 575 580 585 gaa gcc aca cca ttg tcc cag act cga agc cga ggc tcc act aat ctc 2187 Glu Ala Thr Pro Leu Ser Gln Thr Arg Ser Arg Gly Ser Thr Asn Leu 590 595 600 ttt agt aaa tta act tca aaa ctc aca agg agt cgc aat gta tct gct 2235 Phe Ser Lys Leu Thr Ser Lys Leu Thr Arg Ser Arg Asn Val Ser Ala 605 610 615 620 gag caa aaa gat gaa aac aaa gaa gca aag cct cga tcc cta cgc ttc 2283 Glu Gln Lys Asp Glu Asn Lys Glu Ala Lys Pro Arg Ser Leu Arg Phe 625 630 635 acc tgg agc atg aaa acc act agt tca atg gat ccc ggg gac atg atg 2331 Thr Trp Ser Met Lys Thr Thr Ser Ser Met Asp Pro Gly Asp Met Met 640 645 650 cgg gaa atc cgc aaa gtg ttg gac gcc aat aac tgc gac tat gag cag 2379 Arg Glu Ile Arg Lys Val Leu Asp Ala Asn Asn Cys Asp Tyr Glu Gln 655 660 665 agg gag cgc ttc ttg ctc ttc tgc gtc cac gga gat ggg cac gcg gag 2427 Arg Glu Arg Phe Leu Leu Phe Cys Val His Gly Asp Gly His Ala Glu 670 675 680 aac ctc gtg cag tgg gaa atg gaa gtg tgc aag ctg cca aga ctg tct 2475 Asn Leu Val Gln Trp Glu Met Glu Val Cys Lys Leu Pro Arg Leu Ser 685 690 695 700 ctg aac ggg gtc cgg ttt aag cgg ata tcg ggg aca tcc ata gcc ttc 2523 Leu Asn Gly Val Arg Phe Lys Arg Ile Ser Gly Thr Ser Ile Ala Phe 705 710 715 aaa aat att gct tcc aaa att gcc aat gag cta aag ctg taa cccagtgatt 2575 Lys Asn Ile Ala Ser Lys Ile Ala Asn Glu Leu Lys Leu 720 725 atgatgtaaa ttaagtagca agtaaagtgt tttcctgaac actgatggaa atgtatagaa 2635 taatatttag gcaataacgt ctgcatcttc taaatcatga aattaaagtc tgaggacgag 2695 agc 2698 5 20 DNA Artificial Sequence PCR Primer 5 tgaccatgct ggaccagcta 20 6 22 DNA Artificial Sequence PCR Primer 6 tcaccatctg cagtgcttgt ct 22 7 28 DNA Artificial Sequence PCR Probe 7 ccttctgttg tggcgtatcc gaaaagga 28 8 19 DNA Artificial Sequence PCR Primer 8 gaaggtgaag gtcggagtc 19 9 20 DNA Artificial Sequence PCR Primer 9 gaagatggtg atgggatttc 20 10 20 DNA Artificial Sequence PCR Probe 10 caagcttccc gttctcagcc 20 11 2914 DNA H. sapiens CDS (172)...(2313) 11 gacggcccgg gccaggcccg ggatctagaa cggccgtagg gggaagggag ccgccctccc 60 cacggcgcct tttcggaact gccgtggact cgaggacgct ggtcgccggc ctcctagggc 120 tgtgctgttt tgttttgacc ctcgcattgt gcagaattaa agtgcagtaa a atg tcc 177 Met Ser 1 act agg acc cca ttg cca acg gtg aat gaa cga gac act gaa aac cac 225 Thr Arg Thr Pro Leu Pro Thr Val Asn Glu Arg Asp Thr Glu Asn His 5 10 15 acg tca cat gga gat ggg cgt caa gaa gtt acc tct cgt acc agc cgc 273 Thr Ser His Gly Asp Gly Arg Gln Glu Val Thr Ser Arg Thr Ser Arg 20 25 30 tca gga gct cgg tgt aga aac tct ata gcc tcc tgt gca gat gaa caa 321 Ser Gly Ala Arg Cys Arg Asn Ser Ile Ala Ser Cys Ala Asp Glu Gln 35 40 45 50 cct cac atc gga aac tac aga ctg ttg aaa aca atc ggc aag ggg aat 369 Pro His Ile Gly Asn Tyr Arg Leu Leu Lys Thr Ile Gly Lys Gly Asn 55 60 65 ttt gca aaa gta aaa ttg gca aga cat atc ctt aca ggc aga gag gtt 417 Phe Ala Lys Val Lys Leu Ala Arg His Ile Leu Thr Gly Arg Glu Val 70 75 80 gca ata aaa ata att gac aaa act cag ttg aat cca aca agt cta caa 465 Ala Ile Lys Ile Ile Asp Lys Thr Gln Leu Asn Pro Thr Ser Leu Gln 85 90 95 aag ctc ttc aga gaa gta aga ata atg aag att tta aat cat ccc aat 513 Lys Leu Phe Arg Glu Val Arg Ile Met Lys Ile Leu Asn His Pro Asn 100 105 110 ata gtg aag tta ttc gaa gtc att gaa act caa aaa aca ctc tac cta 561 Ile Val Lys Leu Phe Glu Val Ile Glu Thr Gln Lys Thr Leu Tyr Leu 115 120 125 130 atc atg gaa tat gca agt gga ggt aaa gta ttt gac tat ttg gtt gca 609 Ile Met Glu Tyr Ala Ser Gly Gly Lys Val Phe Asp Tyr Leu Val Ala 135 140 145 cat ggc agg atg aag gaa aaa gaa gca aga tct aaa ttt aga cag att 657 His Gly Arg Met Lys Glu Lys Glu Ala Arg Ser Lys Phe Arg Gln Ile 150 155 160 gtg tct gca gtt caa tac tgc cat cag aaa cgg atc gta cat cga gac 705 Val Ser Ala Val Gln Tyr Cys His Gln Lys Arg Ile Val His Arg Asp 165 170 175 ctc aag gct gaa aat cta ttg tta gat gcc gat atg aac att aaa ata 753 Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala Asp Met Asn Ile Lys Ile 180 185 190 gca gat ttc ggt ttt agc aat gaa ttt act gtt ggc ggt aaa ctc gac 801 Ala Asp Phe Gly Phe Ser Asn Glu Phe Thr Val Gly Gly Lys Leu Asp 195 200 205 210 acg ttt tgt ggc agt cct cca tac gca gca cct gag ctc ttc cag ggc 849 Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro Glu Leu Phe Gln Gly 215 220 225 aag aaa tat gac ggg cca gaa gtg gat gtg tgg agt ctg ggg gtc att 897 Lys Lys Tyr Asp Gly Pro Glu Val Asp Val Trp Ser Leu Gly Val Ile 230 235 240 tta tac aca cta gtc agt ggc tca ctt ccc ttt gat ggg caa aac cta 945 Leu Tyr Thr Leu Val Ser Gly Ser Leu Pro Phe Asp Gly Gln Asn Leu 245 250 255 aag gaa ctg aga gag aga gta tta aga ggg aaa tac aga att ccc ttc 993 Lys Glu Leu Arg Glu Arg Val Leu Arg Gly Lys Tyr Arg Ile Pro Phe 260 265 270 tac atg tct aca gac tgt gaa aac ctt ctc aaa cgt ttc ctg gtg cta 1041 Tyr Met Ser Thr Asp Cys Glu Asn Leu Leu Lys Arg Phe Leu Val Leu 275 280 285 290 aat cca att aaa cgc ggc act cta gag caa atc atg aag gac agg tgg 1089 Asn Pro Ile Lys Arg Gly Thr Leu Glu Gln Ile Met Lys Asp Arg Trp 295 300 305 atc aat gca ggg cat gaa gaa gat gaa ctc aaa cca ttt gtt gaa cca 1137 Ile Asn Ala Gly His Glu Glu Asp Glu Leu Lys Pro Phe Val Glu Pro 310 315 320 gag cta gac atc tca gac caa aaa aga ata gat att atg gtg gga atg 1185 Glu Leu Asp Ile Ser Asp Gln Lys Arg Ile Asp Ile Met Val Gly Met 325 330 335 gga tat tca caa gaa gaa att caa gaa tct ctt agt aag atg aaa tac 1233 Gly Tyr Ser Gln Glu Glu Ile Gln Glu Ser Leu Ser Lys Met Lys Tyr 340 345 350 gat gaa atc aca gct aca tat ttg tta ttg ggg aga aaa tct tca gag 1281 Asp Glu Ile Thr Ala Thr Tyr Leu Leu Leu Gly Arg Lys Ser Ser Glu 355 360 365 370 gtt agg ccg agc agt gat ctc aac aac agt act ggc cag tct cct cac 1329 Val Arg Pro Ser Ser Asp Leu Asn Asn Ser Thr Gly Gln Ser Pro His 375 380 385 cac aaa gtg cag aga agt gtt tct tca agc caa aag caa aga cgc tac 1377 His Lys Val Gln Arg Ser Val Ser Ser Ser Gln Lys Gln Arg Arg Tyr 390 395 400 agt gac cat gct gga cca ggt att cct tct gtt gtg gcg tat ccg aaa 1425 Ser Asp His Ala Gly Pro Gly Ile Pro Ser Val Val Ala Tyr Pro Lys 405 410 415 agg agt cag acc agc act gca gat agt gac ctc aaa gaa gat gga att 1473 Arg Ser Gln Thr Ser Thr Ala Asp Ser Asp Leu Lys Glu Asp Gly Ile 420 425 430 tcc tcc cgg aaa tca act ggc agt gct gtt gga gga aag gga att gct 1521 Ser Ser Arg Lys Ser Thr Gly Ser Ala Val Gly Gly Lys Gly Ile Ala 435 440 445 450 cca gcc agt ccc atg ctt ggg aat gca agt aat cct aat aag gcg gat 1569 Pro Ala Ser Pro Met Leu Gly Asn Ala Ser Asn Pro Asn Lys Ala Asp 455 460 465 att cct gaa cgc aag aaa agc tcc act gtc cct agt agt aac aca gca 1617 Ile Pro Glu Arg Lys Lys Ser Ser Thr Val Pro Ser Ser Asn Thr Ala 470 475 480 tct ggt gga atg aca cga cga aat act tat gtt tgc agt gag aga act 1665 Ser Gly Gly Met Thr Arg Arg Asn Thr Tyr Val Cys Ser Glu Arg Thr 485 490 495 aca gat gat aga cac tca gtg att cag aat ggc aaa gaa aac agc act 1713 Thr Asp Asp Arg His Ser Val Ile Gln Asn Gly Lys Glu Asn Ser Thr 500 505 510 att cct gat cag aga act cca gtt gct tca aca cac agt atc agt agt 1761 Ile Pro Asp Gln Arg Thr Pro Val Ala Ser Thr His Ser Ile Ser Ser 515 520 525 530 gca gcc acc cca gat cga atc cgc ttc cca aga ggc act gcc agt cgt 1809 Ala Ala Thr Pro Asp Arg Ile Arg Phe Pro Arg Gly Thr Ala Ser Arg 535 540 545 agc act ttc cac ggc cag ccc cgg gaa cgg cga acc gca aca tat aat 1857 Ser Thr Phe His Gly Gln Pro Arg Glu Arg Arg Thr Ala Thr Tyr Asn 550 555 560 ggc cct cct gcc tct ccc agc ctg tcc cat gaa gcc aca cca ttg tcc 1905 Gly Pro Pro Ala Ser Pro Ser Leu Ser His Glu Ala Thr Pro Leu Ser 565 570 575 cag act cga agc cga ggc tcc act act ctc ttt agt aaa tta act tca 1953 Gln Thr Arg Ser Arg Gly Ser Thr Thr Leu Phe Ser Lys Leu Thr Ser 580 585 590 aaa ctc aca agg agt cgc aat gta tct gct aag caa aaa gat gaa aac 2001 Lys Leu Thr Arg Ser Arg Asn Val Ser Ala Lys Gln Lys Asp Glu Asn 595 600 605 610 aaa gaa gca aag cct cga tcc cta cgc ttc acc tgg agc atg aaa acc 2049 Lys Glu Ala Lys Pro Arg Ser Leu Arg Phe Thr Trp Ser Met Lys Thr 615 620 625 act agt tca atg gat ccc ggg gac atg atg cgg gaa atc cgc aaa gtg 2097 Thr Ser Ser Met Asp Pro Gly Asp Met Met Arg Glu Ile Arg Lys Val 630 635 640 ttg gac gcc aat aac tgc gac tat gag cag agg gag cgc ttc ttg ctc 2145 Leu Asp Ala Asn Asn Cys Asp Tyr Glu Gln Arg Glu Arg Phe Leu Leu 645 650 655 ttc tgc gtc cac gga gat ggg cac gcg gag aac ctc gtg cag tgg gaa 2193 Phe Cys Val His Gly Asp Gly His Ala Glu Asn Leu Val Gln Trp Glu 660 665 670 atg gaa gtg tgc aag ctg cca aga ctg tct ctg aac ggg gtc cgg ttt 2241 Met Glu Val Cys Lys Leu Pro Arg Leu Ser Leu Asn Gly Val Arg Phe 675 680 685 690 aag cgg ata tcg ggg aca tcc ata gcc ttc aaa aat att gct tcc aaa 2289 Lys Arg Ile Ser Gly Thr Ser Ile Ala Phe Lys Asn Ile Ala Ser Lys 695 700 705 att gcc aat gag cta aag ctg taa cccagtgatt atgatgtaaa ttaagtagca 2343 Ile Ala Asn Glu Leu Lys Leu 710 agtaaagtgt tttcctgaac actgatggaa atgtatagaa taatatttag gcaataacgt 2403 ctgcatcttc taaatcatga aattaaagtc tgaggacgag agcacgcctg ggagcgaaag 2463 ctggcctttt ttctacgaat gcactacatt aaagatgtgc aacctatgcg ccccctgccc 2523 tacttccgtt accctgagag tcggcgtgtg gccccatctc catgtgcctc ccgtctgggt 2583 gggtgtgaga gtggacggta tgtgtgtgaa gtggtgtata tggaagcatc tccctacact 2643 ggcagccagt cattactagt acctctgcgg gagatcatcc ggtgctaaaa cattacagtt 2703 gccaaggagg aaaatactga atgactgcta agaattaacc ttaagaccag ttcatagtta 2763 atacaggttt acagttcatg cctgtggttt tgtgtttgtt gttttgtgtt tttttagtgc 2823 aaaaggttta aatttatagt tgtgaacatt gcttgtgtgt gtttttctaa gtagattcac 2883 aagataatta aaaattcact ttttctcagg t 2914 12 3895 DNA H. sapiens CDS (1504)...(3762) 12 ctgcaggaat tccgatcctt ccgcaggttc acctacggaa accttgttac gacttttact 60 tcctctagat agtcaagttc gaccgtcttc tcagcgctcc gccagggccg tgggccgacc 120 ccggcggggc cgatccgagg gcctcactaa accatccaat cggtagtagc gacgggcggt 180 gtgtacaaag ggcagggact taatcaacgc aagcttatga cccgcactta ctgggaattc 240 ctcgttcatg gggaataatt gcaatccccg atccccatca cgaatggggt tcaacgggtt 300 acccgcgcct gccggcgtag ggtaggcaca cgctgagcca gtcagtgtag cgcgcgtgca 360 gccccggaca tctaagggca tcacagacct gttattgctc aatctcgggt ggctgaacgc 420 cacttgtccc tctaagaagt tgggggacgc cgaccgctcg ggggtcgcgt aactagttag 480 catgccagag tctcgttcgt tatcggaatt aaccagacaa atcgctccac caactaagaa 540 cggccatgca ccaccaccca cggaatcgag aaagagctat caatctgtca atcctgtccg 600 tgtccgggcc gggtgaggtt tcccgtgttg agtcaaatta agccgcaggc tccactcctg 660 gtggtgccct tccgtcaatt cctttaagtt tcagctttgc aaccatactc cccccggaac 720 ccaaagactt tggtttcccg gaagctgccc ggcgggtcat gggaataacg ccgccgcatc 780 gccggtcggc atcgtttatg gtcggaacta cgacggtatc tgatcgtctt cgaacctccg 840 actttcgttc ttgattaatg aaaacattct tggcaaatgc tttcgctctg gtccgtcttg 900 cgccggtcca agaatttcgg aattccgcag cggcggccag cagggcggag gctgaggcag 960 caagctcgct agagagggag aagcagtcgg gcgcaggcgc ctcctccgca gcccgctcca 1020 tggtcggcgc ccacagcccg cggcggcctg tcttgcgctc cacttccttc acatcctcct 1080 ccgcctcctc gttttcaggc gccgccggcg gcgctgtgtg gaggcccgcg agctgaaatt 1140 cgcggtgcga cgggagggag tggagaagga ggtgaggggg cccaggatcg cggggcgccc 1200 tgaggcaagg ggacgccggc gggccgaagc gcagcccgcc gcccgcaggc tcggctccgc 1260 cactgccgcc ctcccggtct cctcgcctcg gccgccgagg cagggagaga atgagccccg 1320 ggacccgccg ggggacggcc cgggccaggc ccgggatcta gacggccgta gggggaaggg 1380 agccgccctc cccacggcgc cttttcggaa ctgccgtgga ctcgaggacg ctggtcgccg 1440 gcctcctagg gctgtgctgt tttgttttga ccctcgcatt gtgcagaatt aaagtgcagt 1500 aaa atg tcc act agg acc cca ttg cca acg gtg aat gaa cga gac act 1548 Met Ser Thr Arg Thr Pro Leu Pro Thr Val Asn Glu Arg Asp Thr 1 5 10 15 gaa aac cac acg tca cat gga gat ggg cgt caa gaa gtt acc tct cgt 1596 Glu Asn His Thr Ser His Gly Asp Gly Arg Gln Glu Val Thr Ser Arg 20 25 30 acc agc cgc tca gga gct cgg tgt aga aac tct ata gcc tcc tgt gca 1644 Thr Ser Arg Ser Gly Ala Arg Cys Arg Asn Ser Ile Ala Ser Cys Ala 35 40 45 gat gaa caa cct cac atc gga aac tac aga ctg ttg aaa aca atc ggc 1692 Asp Glu Gln Pro His Ile Gly Asn Tyr Arg Leu Leu Lys Thr Ile Gly 50 55 60 aag ggg aat ttt gca aaa gta aaa ttg gca aga cat atc ctt aca ggc 1740 Lys Gly Asn Phe Ala Lys Val Lys Leu Ala Arg His Ile Leu Thr Gly 65 70 75 aga gag gtt gca ata aaa ata att gac aaa act cag ttg aat cca aca 1788 Arg Glu Val Ala Ile Lys Ile Ile Asp Lys Thr Gln Leu Asn Pro Thr 80 85 90 95 agt cta caa aag ctc ttc aga gaa gta aga ata atg aag att tta aat 1836 Ser Leu Gln Lys Leu Phe Arg Glu Val Arg Ile Met Lys Ile Leu Asn 100 105 110 cat ccc aat ata gtg aag tta ttc gaa gtc att gaa act gaa aaa aca 1884 His Pro Asn Ile Val Lys Leu Phe Glu Val Ile Glu Thr Glu Lys Thr 115 120 125 ctc tac cta atc atg gaa tat gca agt gga ggt gaa gta ttt gac tat 1932 Leu Tyr Leu Ile Met Glu Tyr Ala Ser Gly Gly Glu Val Phe Asp Tyr 130 135 140 ttg gtt gca cat ggc aag atg aag gaa aaa gaa gca aga tct aaa ttt 1980 Leu Val Ala His Gly Lys Met Lys Glu Lys Glu Ala Arg Ser Lys Phe 145 150 155 aga cag ggt tgt caa gct gga cag act att aaa gtt caa gtc tcc ttt 2028 Arg Gln Gly Cys Gln Ala Gly Gln Thr Ile Lys Val Gln Val Ser Phe 160 165 170 175 gat ttg ctt agt ctg atg ttt aca ttt att gtg tct gca gtt caa tac 2076 Asp Leu Leu Ser Leu Met Phe Thr Phe Ile Val Ser Ala Val Gln Tyr 180 185 190 tgc cat cag aaa cgg atc gta cat cga gac ctc aag gct gaa aat cta 2124 Cys His Gln Lys Arg Ile Val His Arg Asp Leu Lys Ala Glu Asn Leu 195 200 205 ttg tta gat gcc gat atg aac att aaa ata gca gat ttc ggt ttt agc 2172 Leu Leu Asp Ala Asp Met Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser 210 215 220 aat gaa ttt act gtt ggc ggt aaa ctc gac acg ttt tgt ggc agt cct 2220 Asn Glu Phe Thr Val Gly Gly Lys Leu Asp Thr Phe Cys Gly Ser Pro 225 230 235 cca tac gca gca cct gag ctc ttc cag ggc aag aaa tat gac ggg cca 2268 Pro Tyr Ala Ala Pro Glu Leu Phe Gln Gly Lys Lys Tyr Asp Gly Pro 240 245 250 255 gaa gtg gat gtg tgg agt ctg ggg gtc att tta tac aca cta gtc agt 2316 Glu Val Asp Val Trp Ser Leu Gly Val Ile Leu Tyr Thr Leu Val Ser 260 265 270 ggc tca ctt ccc ttt gat ggg caa aac cta aag gaa ctg aga gag aga 2364 Gly Ser Leu Pro Phe Asp Gly Gln Asn Leu Lys Glu Leu Arg Glu Arg 275 280 285 gta tta aga ggg aaa tac aga att ccc ttc tac atg tct aca gac tgt 2412 Val Leu Arg Gly Lys Tyr Arg Ile Pro Phe Tyr Met Ser Thr Asp Cys 290 295 300 gaa aac ctt ctc aaa cgt ttc ctg gtg cta aat cca att aaa cgc ggc 2460 Glu Asn Leu Leu Lys Arg Phe Leu Val Leu Asn Pro Ile Lys Arg Gly 305 310 315 act cta gag caa atc atg aag gac agg tgg atc aat gca ggg cat gaa 2508 Thr Leu Glu Gln Ile Met Lys Asp Arg Trp Ile Asn Ala Gly His Glu 320 325 330 335 gaa gat gaa ctc aaa cca ttt gtt gaa cca gag cta gac atc tca gac 2556 Glu Asp Glu Leu Lys Pro Phe Val Glu Pro Glu Leu Asp Ile Ser Asp 340 345 350 caa aaa aga ata gat att atg gtg gga atg gga tat tca caa gaa gaa 2604 Gln Lys Arg Ile Asp Ile Met Val Gly Met Gly Tyr Ser Gln Glu Glu 355 360 365 att caa gaa tct ctt agt aag atg aaa tac gat gaa atc aca gct aca 2652 Ile Gln Glu Ser Leu Ser Lys Met Lys Tyr Asp Glu Ile Thr Ala Thr 370 375 380 tat ttg tta ttg ggg aga aaa tct tca gag ctg gat gct agt gat tcc 2700 Tyr Leu Leu Leu Gly Arg Lys Ser Ser Glu Leu Asp Ala Ser Asp Ser 385 390 395 agt tct agc agc aat ctt tca ctt gct aag gtt agg ccg agc agt gat 2748 Ser Ser Ser Ser Asn Leu Ser Leu Ala Lys Val Arg Pro Ser Ser Asp 400 405 410 415 ctc aac aac agt act ggc cag tct cct cac cac aaa gtg cag aga agt 2796 Leu Asn Asn Ser Thr Gly Gln Ser Pro His His Lys Val Gln Arg Ser 420 425 430 gtt tct tca agc caa aag caa aga cgc tac agt gac cat gct gga cca 2844 Val Ser Ser Ser Gln Lys Gln Arg Arg Tyr Ser Asp His Ala Gly Pro 435 440 445 gct att cct tct gtt gtg gcg tat ccg aaa agg agt cag aca agc act 2892 Ala Ile Pro Ser Val Val Ala Tyr Pro Lys Arg Ser Gln Thr Ser Thr 450 455 460 gca gat ggt gac ctc aaa gaa gat gga att tcc tcc cgg aaa tca agt 2940 Ala Asp Gly Asp Leu Lys Glu Asp Gly Ile Ser Ser Arg Lys Ser Ser 465 470 475 ggc agt gct gtt gga gga aag gga att gct cca gcc agt ccc atg ctt 2988 Gly Ser Ala Val Gly Gly Lys Gly Ile Ala Pro Ala Ser Pro Met Leu 480 485 490 495 ggg aat gca agt aat cct aat aag gcg gat att cct gaa cgc aag aaa 3036 Gly Asn Ala Ser Asn Pro Asn Lys Ala Asp Ile Pro Glu Arg Lys Lys 500 505 510 agc tcc act gtc cct agt agt aac aca gca tct ggt gga atg aca cga 3084 Ser Ser Thr Val Pro Ser Ser Asn Thr Ala Ser Gly Gly Met Thr Arg 515 520 525 cga aat act tat gtt tgc agt gag aga act aca gct gat aga cac tca 3132 Arg Asn Thr Tyr Val Cys Ser Glu Arg Thr Thr Ala Asp Arg His Ser 530 535 540 gtg att cag aat ggc aaa gaa aac agc act att cct gat cag aga act 3180 Val Ile Gln Asn Gly Lys Glu Asn Ser Thr Ile Pro Asp Gln Arg Thr 545 550 555 cca gtt gct tca aca cac agt atc agt agt gca gcc acc cca gat cga 3228 Pro Val Ala Ser Thr His Ser Ile Ser Ser Ala Ala Thr Pro Asp Arg 560 565 570 575 atc cgc ttc cca aga ggc act gcc agt cgt agc act ttc cac ggc cag 3276 Ile Arg Phe Pro Arg Gly Thr Ala Ser Arg Ser Thr Phe His Gly Gln 580 585 590 ccc cgg gaa cgg cga acc gca aca tat aat ggc cct cct gcc tct ccc 3324 Pro Arg Glu Arg Arg Thr Ala Thr Tyr Asn Gly Pro Pro Ala Ser Pro 595 600 605 agc ctg tcc cat gaa gcc aca cca ttg tcc cag act cga agc cga ggc 3372 Ser Leu Ser His Glu Ala Thr Pro Leu Ser Gln Thr Arg Ser Arg Gly 610 615 620 tcc act aat ctc ttt agt aaa tta act tca aaa ctc aca agg agt cgc 3420 Ser Thr Asn Leu Phe Ser Lys Leu Thr Ser Lys Leu Thr Arg Ser Arg 625 630 635 aat gta tct gct gag caa aaa gat gaa aac aaa gaa gca aag cct cga 3468 Asn Val Ser Ala Glu Gln Lys Asp Glu Asn Lys Glu Ala Lys Pro Arg 640 645 650 655 tcc cta cgc ttc acc tgg agc atg aaa acc act agt tca atg gat ccc 3516 Ser Leu Arg Phe Thr Trp Ser Met Lys Thr Thr Ser Ser Met Asp Pro 660 665 670 ggg gac atg atg cgg gaa atc cgc aaa gtg ttg gac gcc aat aac tgc 3564 Gly Asp Met Met Arg Glu Ile Arg Lys Val Leu Asp Ala Asn Asn Cys 675 680 685 gac tat gag cag agg gag cgc ttc ttg ctc ttc tgc gtc cac gga gat 3612 Asp Tyr Glu Gln Arg Glu Arg Phe Leu Leu Phe Cys Val His Gly Asp 690 695 700 ggg cac gcg gag aac ctc gtg cag tgg gaa atg gaa gtg tgc aag ctg 3660 Gly His Ala Glu Asn Leu Val Gln Trp Glu Met Glu Val Cys Lys Leu 705 710 715 cca aga ctg tct ctg aac ggg gtc cgg ttt aag cgg ata tcg ggg aca 3708 Pro Arg Leu Ser Leu Asn Gly Val Arg Phe Lys Arg Ile Ser Gly Thr 720 725 730 735 tcc ata gcc ttc aaa aat att gct tcc aaa att gcc aat gag cta aag 3756 Ser Ile Ala Phe Lys Asn Ile Ala Ser Lys Ile Ala Asn Glu Leu Lys 740 745 750 ctg taa cccagtgatt atgatgtaaa ttaagtagca agtaaagtgt tttcctgaac 3812 actgatggaa atgtatagaa taatatttag gcaataacgt ctgcatcttc taaatcatga 3872 aattaaagtc tgaggacgag agc 3895 13 506 DNA H. sapiens 13 ggaggtttgt gtcgtgtttg gttaactaaa cctaaaggtg acttactcgt tttctttcct 60 ctgtacctct ccaaaggaac tgagagagag agtattaaga gggaaataca gaattccctt 120 ctacatgtct acagactgtg aaaaccttct caaacgtttc ctggtgctaa atccaattaa 180 acgcggcact ctagaggtaa tcatgtaggt ggaaacaagc agtaactttg gagagtcttt 240 agagtgacct tagatcttgg cttgatttgt atgccatact ggatatatcc tgcggctttt 300 taagcaagaa tggaaacatt aaaaaatatt tttggagttt atgctttgaa cgatagtcaa 360 tgaaatgttg aaaataaatt ttggtaaata ttacggttat cagaatattt cattttactc 420 tgctaatgaa cagtttacct tttttagcaa atcatgaagg acaggtggat caatgcaggg 480 catgaagaag atgaactcaa accatt 506 14 506 DNA H. sapiens 14 ggaggtttgt gtcgtgtttg gttaactaaa cctaaaggtg acttactcgt tttctttcct 60 ctgtacctct ccaaaggaac tgagagagag agtattaaga gggaaataca gaattccctt 120 ctacatgtct acagactgtg aaaaccttct caaacgtttc ctggtgctaa atccaattaa 180 acgcggcact ctagaggtaa tcatgtaggt ggaaacaagc agtaactttg gagagtcttt 240 agagtgacct tagatcttgg cttgatttgt atgccatact ggatatatcc tgcggctttt 300 taagcaagaa tggaaacatt aaaaaatatt tttggagttt atgctttgaa cgatagtcaa 360 tgaaatgttg aaaataaatt ttggtaaata ttacggttat cagaatattt cattttactc 420 tgctaatgaa cagtttacct tttttagcaa atcatgaagg acaggtggat caatgcaggg 480 catgaagaag atgaactcaa accatt 506 15 119501 DNA Homo sapiens 15 ggcgcctgct gccctcaggg tccgcgccca gcccgcagct gctcagatcc ggagacggga 60 aggtttgttg gcgagaacct gactcccggg tcacagttaa ggatgcaaga gcccggcgcc 120 ttcccgtagc cccggccctg tcattaatta atgctggggc tccattcggt gcagcgcagt 180 cccagggatg caaccgcaac ttttgcgcac aataggctct cgatctgtaa tccagccaac 240 ccaggcctgt agtgtgtaaa tgccaactca gcgggagggc tctggctgtc gcccagagcc 300 gtttctcggc tctttcgcgg ttgccggcgc gctcgggaca ggaggaaccc gcagcccgcg 360 ggagtcagcg gagacccgac cagcactatc cagccctctg caccgccccc gcggcgaggt 420 ctggaccaag tcgcccctag caacaacagc cgggccggct ttctcaggcc atgctgattg 480 gcgggactcc gggtggcggc ctgtcgtcac ttccggcagc cggaggcagc agaggaagcc 540 gaggggcggc catcttggct ccgtgaggct ctgaggtgcc ggggtgcggc ggcggcagcg 600 gcggccagca gggcggaggc tgaggcagca agctcgctag agagggagaa gcagtcgggc 660 gcaggcgcct cctccgcagc ccgctccatg gtcggcgccc acagcccgcg gcggcctgtc 720 ttgcgctcca cttccttcac atcctcctcc gcctcctcgt tttcaggcgc cgccggcggc 780 gctgtgtgga ggcccgcgag ctgaaattcg cggtgcgacg ggagggagtg gagaaggagg 840 tgagggggcc caggatcgcg gggcgccctg aggcaagggg acgccggcgg gccgaagcgc 900 agcccgccgc ccgcaggctc ggctccgcca ctgccgccct cccggtctcc tcgcctcggc 960 cgccgaggca gggagagaat gagccccggg acccgccggg ggacggcccg ggccaggccc 1020 gggatctaga cggccgtagg gggaagggag ccgccctccc cacggcgcct tttcggaact 1080 gccgtggact cgaggacgct ggtcgccggc ctcctagggc tgtgctgttt tgttttgacc 1140 ctcgcattgt gcagaattaa agtgcagtaa aatgtccact aggaccccat tgccaacggt 1200 gaatgaacga gacactgaaa acgtaagtaa cctgggcgtt gtagttggcg gaccttcggg 1260 gtggcattcg tgctcctcgg gcagtgcctt gcagtcgggt gttcccccca gccgaacgct 1320 ctggaaatag agggcaggcc gtagtcaccc aggaggcagc caggtcggac cgtttcctcc 1380 agaagtctgc cgtgtcccgc tgttcgcggg cgggtctgcg aagtacatcg attatgccgg 1440 cagtctagtc ggttaataaa gcccaggagt tgcagcgtta cggatcggtg ctttgagagg 1500 ccaggttgcc gcgcaatgga ttgtgcaaac gtgtgtgtca gatcactgca gtggtcagtg 1560 cttattttag ccgaactctg cttttaactt ggtcaggcag ttcttgagag actgtcacat 1620 taataacata tgttaccggt gctgattaag agtaatcgat tgggtcaagt gggcggtctc 1680 gttctttaag atggtctgga actaaattta atcacagtca ctaagtgtcc actgagggat 1740 ctttgcggtc cccttaggaa ttccaaaaca ctagcgcgca gcctttatta aggcagtttc 1800 ctttccatac aaatcttttt tgaaggagtt ggttaaacat tttgaaaagc agatgtatag 1860 tgaagctctg ctctgccact cactgcctag ctgagggaca cccgctgggg gcttcgctgt 1920 cctccttcct aaaaagagat tggcttagat gagctcttag gtccctttca gcgctcacag 1980 gctatggttt tataaaagga acctttgatt ttgttcatgt gaaactacaa aatgccagga 2040 acagcattgc tagagaatga gaacttgtag tagaaattcc attgaagaaa cgtttgactc 2100 tgttcgtttt ctaactctgt tctcctctaa cactgggttc aaaagtgttt caatctagtt 2160 gttgactata ttctgtaaaa catctagtaa ataatttgta aagaaacttc actggccttt 2220 cattaaaagt gttatgaggt aattgctggc acgaaatgtc ttctcttagg gaatgcttcc 2280 ttgctttgag tgttttcatg ctttcagtaa tgtcctgaga tgttgtggcc tagtggagtt 2340 aatgctttca ttttgatctt caaaaatgcc gcttgtttgc tggatagctg tttgataatt 2400 taatccgtaa acgtaaacca tagcctaaag catccctgta gcatattagt atacaatatt 2460 taatgctctg tggccctgag taagtagaac agtgatgtac ctttctccac ctgagataga 2520 ttagaaaatt atataaagag tgagtaatag aaactcttag actaggtgag tcagggaact 2580 aatttcctta tagtcctttt aaagaaacct tatttatttg tttatttatt tatttattta 2640 cttatttatt ttttgagaca gaccgagact gtctcccagg ctggagtgca gtggtgcgat 2700 cttggctcac tgcagcctcc acctcccagg ctgaagtgat tatcatgcct cagcctcccg 2760 agtagctggg attactggcg tgcaccacca cgcccggcta atttttgtat ttttagtaga 2820 gacagggttt caccatgttg gccaggctgg tctagaactc ctggcctcat gtgatccacc 2880 cgcctcggct ccccaaagtg ctgagattac aggcatgagc cagtgtgccc aactgattag 2940 ttttgatcat tgatcttcac tcagagaagc aaaaccttac attgaaccag agtaggagct 3000 ccatgctttc taaactgaag ccaaaaacag tcatagcaga tttgtgataa tgaaggattt 3060 caaatgggtc ttttttcttt ctttcttttt tgagatggag tctggctctg tctcccaggc 3120 tggagtgccg tggcatgatc tcggctcact gcaacctccg cctcccgggt tcaagtgatt 3180 ctcctgcctc agcctcctga gtagccagga ttacaggcat gtgccaccac tcccggctaa 3240 ttttgtattt ttagtagaga cggggtttct ccatgttggt cgtgctggtc tcaaactccc 3300 gacttcaggt gatcctcccg cgtcagcctc ccaaagtgct gggattacag gcgtgagcca 3360 ccacgcccag ccatgtagtg tatttaaaat gaatttttga ctttttttaa aaatcaagtt 3420 tatcacacat cgttgcatta atttaccatg ccctgttaca tttttaactc ttgcattggc 3480 atgttctgga aggagctgtg tagctttcac agtgtggagc ccttgtgtca gtgttataac 3540 tcaggtatag tccatattaa ttacctacat tactgcactg ctaagtatta gacttcctgc 3600 caattgagtg gtgaatgtac aagaatgaat gggagctctc attctgttga gaaccttttt 3660 tcttaaagtt aggtttgttg agatgtaatg ttcatctgtt ttaagtatac agtttggtga 3720 attttgacaa gcgtatagtt atgtaaccgc tatcacaatc aagatgtaga acacttccat 3780 tgccagaaag gtcccttatg gccctgtata gtcagtgccc tctccctttc ttagcccctg 3840 ggtaaccact gatctgcttt ctgtccctgt agttttgcct tttctaggat gctatgtaaa 3900 tggagtcaca gtgtaaataa tcttttgtgt cttctttcac ttagagtagt gcttttgaga 3960 tttatttgaa tgtgtatcag tatcagtagt tcattccttt ttattgctga gtaagtattt 4020 ccttgtttgg atgtgccaca atttttttta atccactcac cagtgatgga catttttggc 4080 tattgtgact agaacagttt tcttttagta ttctgtaaaa atacttttta ttgtgcttac 4140 cccaggaatc ttatttaaat tttaagtaat tgtatataac tgtgataaga atgtcgctta 4200 ttagatcaag actaagaaaa ggcaggccgg gtgtggtggc tcatacctgt aatcccagca 4260 ctttgagagg ctgaggctgg cggatcacga ggtcaggaga tcgagaccat cctggctaac 4320 atggtgaaac cctgtctcta ctaaaaatac gaaaaattag ccgggcgtgg tggcaggcac 4380 ctgtagtcct agctactcgg gaggctgagg caggagaatg gcatgaacct gggaggcaga 4440 gcttgcagtg agccgagatt gggccaccac attccagcct gggcgacaga gccagactcc 4500 atctcaaaaa aaaaaaaaaa ttagccaggt gcggtggtgc atgcctgtaa ttccagctac 4560 tcaggaggct gacgtgggag aatcacttga acacaagagg tgaaggttgc agtgagccga 4620 tatcatgcca ctgcactgca gcctgagtga cagagtaaga ctctgtctcc aaaaaaaaaa 4680 aaaaaaaaaa gcagacaact tgcaacatta tcttcatcct tgtacactaa tttgtttttg 4740 tttgtttgat ttccaaatat taatgtccac ccttagtgaa tgggtatata aaagtttact 4800 gttggccagg tgcagtggct cacgcctgta atcccagcac tttgggaggc cggggcaggt 4860 ggatcacctg aggtcaggag ttcgagacca gcctgaccaa gatggcaaaa ccctgtctct 4920 actaaaaata caaaaattag ccgggtgcgg tggcgggcac ctgtaatccc agctacttgg 4980 gaggctgagg ctggagaatc gcttgaaccc tggagacgga gagtgcagtg agctgagatc 5040 aagccattgc cctccagcct gggcgacaga gggatactcc gtctcaaaaa aaaaaaaaaa 5100 aaaaaaaagg ccgggcgcgg tggctcacac ctgtaatccc agcactctgg gaggccgagg 5160 cgggcagatc acgaggtcag gagatcgaga ccatcctggc taacacggtg aaaccctgtg 5220 tctaccaaaa atataaaaaa ttagccgggc gtggtggcag gtgcctgtag tcccagctac 5280 tcgggaggct gaggcaggag aatggcgtga tcccgggagg cggagcttgc agtgagccaa 5340 gatggcgcca ctgcactcca gcctgggcga cagagtgaga ctccatctca aaaaaaaagt 5400 tcactgttgc tgcatcgtgt tatagctcaa aagaactact caatctgatc cagtgaggtt 5460 gctgagactc agagaggtga caaaggctgc tagtaatgac agaagaccac taactattta 5520 gtagcaaagc ctctactggg ccccagatct ctagcttagg ctccacaagg agatgtagtt 5580 gtacctgtac atagagtatt tctaaaactt ttttttaagc ttgtagatca ggggtgtcca 5640 atcttttggc ttccccgggc cacattggaa gaattgtctt gggccacaga taaaatacac 5700 taacactaac aatagctgat aagctttaaa aaaactgcaa aaaaaatctc acagtgtttt 5760 aacaaagttt atgaatttgt gttgggtcac tttcaaagct gtggtgggct gcatgcagcc 5820 ctagggctgt gggttggaca agcttgctgt agattttatt ttgattttga gacagtatct 5880 cgctctgtca cccaggctgg agtgcagtgg agcaatcaca gcttgctgta accttgacct 5940 cttgggctca agcaatcctt ccgactcagc ctcctgagta gctgggacta caggcgtgcc 6000 ccaccacatg catggctaat ttttaatttt ttcgtagaga tgagatctcc ctgtattgcc 6060 caaggtggtc tctaactcct gggctcaagc aggcctcctg tcccagcctc ccaaagcact 6120 ggaattacag gtgtgagcca ccacactcag tcaaaactat tttttaaaat attttattat 6180 aggaaaattt aaaaaaatag aatgttattt atcaatgctt ctcagccaca gggtgatttt 6240 gccctctagt ggacatttga caatgtctaa agacatcttt ggttatcaca gctggagaaa 6300 ggtttggctg accaggaatg ctactaaaca ttttacagta cgtaggacag ccttccacag 6360 caaagaatta cccggcctaa aatgtcagta gtgccagcgt tgagaaaccc tggtatgtac 6420 ttagcccccc atgtactcaa cagcgcagct cagtggccaa tcttatttca tttataaatg 6480 aagaagcaaa tgcagtatca tttcctctct aaagctttta gaagagataa agcctgtttt 6540 aagaaaaaag caatactttt atcatatttt cttaatatca aatatgcagt atttaaattt 6600 tctcacattt ctaattttta aactgttcaa attagaatcc agcttgaatt catacattat 6660 agttggctga tatgtcaaat atgctttttt ttttttttta attgagatgg agtcttgctc 6720 tgtcgcccag gttggagtgc agtggtgcaa tctcggctca ctgccaacct ctgccgcccg 6780 ggttcaagcg attgtcgtgc gcagcctccc gagtagctgg gattacaggc tcccgccacc 6840 atgcctggct attttttttt tttttttttt ttttttttta gtagagatgg ggtttcgcca 6900 tgttggccag gctgttctca aactcctgac ctcaggtgat ccgcccgcct tggcctccca 6960 aagtgctggg attacaggcg tgagccactg tacctggtca aaatatgctt tttaaaggca 7020 accaaacagt ttgcatttca gtttatgttc tatagttata tggagccacg gatacatcct 7080 ttttctatac tggcaaaatg agaaacatat ttttcacctg aatttcaaag gatatgacac 7140 ttcgtaagcc tggtaatgtt attggccaaa gtaccagagg cccagggctt tcccagttat 7200 ttaacagtta ctgatatgtg tgcatcatgt acttattatt agttaatgca aatagtcccc 7260 caaatttctg acctctgtga aactgatgat gctgttgtct agtcagtcat cggttatgtg 7320 agactgaatt aacctattca gcaagtgtga atgcctaccg tgtgtatgat actgtgctag 7380 gtgctgtaga ggccatatac ctgggacaca tagctttcct gctctttagg atttcctgct 7440 ccttgcagga caaagcagcc tgagctttat gaaactagtc tgtaactctc aaacttaagc 7500 aaacattaga atcacctgaa gggctttaga accgtgatca ctgggcccta cccctagacc 7560 ttgattttac ccgtagaatt ggaatttcta acaagttgcc aggtgatgtt gatgctccag 7620 gtccggggac cactcttaag aatcactata gcaggaattt ctgatgttta tttattaaga 7680 aaaaaaaatc actgccttag tgtgcagtga aggcttgcta actaaactat actcctttta 7740 gattgtactg tgactatcaa aggaagattt ccagaccatt taattgagtt ttcaggttcc 7800 atgagtttaa aaaaaactgt ttattaaaaa ttgtggttct ggaaaataac aatagttgcc 7860 atttggtgat cacttaactg tataccagcc agcaatactt tgtagagtat ctcatttaaa 7920 atatgtaata tggttttatt taattagctt tttttttttt taaatttcct gaggcggagt 7980 ctcacactgt tacctgggct ggagtgcagt ggcacgatct cgctcgctgc aacctccgcc 8040 tcccgggttc aagcgattct cctgcctcag cctcccaggt agctaggatt acaggcagcc 8100 gccaccacac ccagctaatt tttaaatttt attttatttt ttgagacaga gtttcactct 8160 tgttgcccgg gctggagtgc agtggcatga tcttggctca ctgcaacctc cgcctcctgg 8220 cttcaagcaa ttctgcctca gcctcccgag tagctgggat tacaggtgcc tgttaccatg 8280 cccagctgat ttttttgtat tttcggtaga gactgggttt caccatgttg gcaggctgat 8340 ctggaactcc tgacctcatg atcctcccac ttccctcctc ggcctcccaa agtgctggaa 8400 ttacaggcgt gagccactgt gccccggctt aatcagcttg tcaacttcta agcacaacct 8460 gttgaatcaa gctgtttggg ccttatcaga aaaaagttgg cctatagttt cagctactca 8520 ggaggctaag gccagaggat cacttgaggc caggagttcc agtcgacctg gacaacatgg 8580 tgaaactatg tatcttaaga aagaaaaaaa aaagttgatc tctgaagaat tcatgaaaat 8640 atttagtcag ttttcaggga aagatcttat agacttcaat ttgagcatcc ttatcttatg 8700 tatgtaaatg aggtaaaggt tgaaatttgc agtaaataga attttggtca tttaagttat 8760 gtagctcatg atatatacat tgtataacta tttagactta catggtgcat gatttacatt 8820 gtaattacat agaggcttat acagtctaat aaaaattaat attaactatt tgattgcaaa 8880 gaggattgtt ctgcatttta ttactttttt tttttttttg agacagagtt tcactcttgt 8940 tgccaggctg gagtgcaatc tcgtgacctt ggctcactgc aacctccgcc tgccagccta 9000 agtagctggg gttacaggca cacaccacca tgcccagcta attttttgta tttttagtag 9060 ggatggggtt tcaccatgtt agccaggctg gtctcaaact cctcaccttg ggttatccgc 9120 ctgccttggc ctcccaaagt gctgggatta caggcttgag ccactgcgct cggcctgcat 9180 tttattattc taagatatag ccaaatttta tgcctggaat tttttcctgg gctggaacaa 9240 attcctagta agagtcaggg aatattccgt gacttgtggc tttggttaaa aaagaaaaag 9300 gagtcaggga atgcagtacc ctctgatcac cactgggagg actaaagcag aaaattcatt 9360 tgtctcatat ccattttcaa atttcaaatt aaacatgaag gagaaaataa atctcttacc 9420 tgggaagctt taatgtcctg tctcacacaa gaagttaaag gttgtgcttg gagttggtct 9480 gcaaaagtaa taaggaattt gtactcaccc gtctgcacct gcttgtacag aacaattgga 9540 agggcagaag ttggctttga aagaaaaggc ctttttttgg aggtgggagg ggcatccaat 9600 tttgtcttca gccgagagtt cgctagcact acactctagc cactcaagct gctcaactat 9660 ggttggtctt gtgtacttct gtgtgcacta gataagacat tgtttctgtg ggttatgtgg 9720 gaccttgaag ttcagtgttg aagctcaaac aaagcaggtg ttttttctgt atttcttgtt 9780 gacaggcatt tgctttttgt tggacctaaa tattgtgggg tttgtgtatg gacatttgac 9840 acaaaaatct gtattggtag aatttaatat aaaatcagag aagttgaatt cacaggtttt 9900 gttcatagaa ttttacataa ctctagtctt ttttcttttt ttgtgatgga gtcttgctct 9960 gttgcccagg ctggagtgca gtggcatgat cttggctcac tgcaacctct gcctcctggg 10020 ttcaagtgat tgtcctgcct cagcctcccg agtagctggg attacaggca cccgccacca 10080 cgcctggcta atttttgtat ttttagtaga aacggggttt caccatggcc aggctggtct 10140 tgaactcctg acctcaagtg atccacctgc ctcggcctcc caaagtgcta ggattatagg 10200 catgagccac agcgcctggc cacaatctta cactactttc ctgaagggat gtgtgatgga 10260 aagagtctgg gcttgaaagt ggaagacaaa aatgagagtc taagactgat cgagataaag 10320 attctgaaga ggtagaatgg acctcataag atggagcaaa aaagaaggaa caacatataa 10380 aaggagaaat tagaatttag agttatagac tggaatttga aactagtctt gagatttaac 10440 tgtggacaaa ttcactcggc tgtgcctcat ggtttttttt tttttaaatc aaaagagaat 10500 aacatcattc aagttgtgtc ttggtttttt tgtttgttta cttagatttt tatgacttca 10560 ctttctcaat ttcctctgtg gccagattca tcaggcattc agtcactttc atctctgtca 10620 ggctggcctt gtgttctgtc tgtctgttct gctgcatagt tgtcatcctg agatctccct 10680 tcacccatat cctagggatt ccctttgcct ttcctgttcg atccattttc ttccccttgg 10740 ttcactccca ggtttcattg cagcagttcc tccagtgatt ttctgagaaa gttgctcaga 10800 ggtaaatttt taaaatcctt tcatatctgc aaacatcttt attctgttct catacttgat 10860 tgatattttg gctgcatata gaattctaga ctgaatgccc tttttcctca gaattttgtt 10920 ttctgtctta tatagcatct gacactccga tttggagaga gagaatgcta ttttgagtct 10980 tgcttatttt tctgtttgtt taaggctgat tggaagtttt gtgcatttgg atggatcttg 11040 aatacaacag tgggacacac tgttatttct ttgtggtgct cttgatacca atatatttag 11100 tcttatcact tggcctaggc aagcacggaa tgcccatgtt ccagggagct gagtcgggag 11160 agggcagaag gaaagatctg tggctttctg ctttctctgc ttgttttcag tgtccctgcc 11220 ctcagttgtg ccctgtgtcc ccaagcccag agactttcca ttttgccctt tctaaagaac 11280 ctccagtttt ttgcctgagt gggagaggag acccgggggt ctgttattta acagactgat 11340 agatcttctt gtttcaggct tacttcactc acacttctag aggtacttgt actaccagtt 11400 cctgaacctt ttgagagttc tataatacaa atcaggattt tccaatatct ccatagctgg 11460 cttaggatta atctttctta tatgtgttaa gtcattttcc ttcttttttt agtttctgaa 11520 agtatatctg ttaccacttt tattttcttt gtcttgtggc ttttttcctt atgccttata 11580 tttttttcct ttactttcat attaatggag tttggagagg gaggggaagt aaatgcagcc 11640 atgatttata accagacttg gattaggtgt ttacatatgt caatacatgt ctttaaaatt 11700 tagctagtac ttgttattat aaataacaaa ctaagattga taggcttctt gaaaaattgg 11760 cggtaaattt ggctaatggt gggttgttgt agtagagtag cgtttcccct tttcacccaa 11820 gctttgttac ctcttcttgg gtggtgcctg gcctgagatg tccttcatct ctcataagaa 11880 ctctcctact tgccctttga ggactccatc ccttcacatc ttggggtacc ttttttcctt 11940 catctcaccc aaatgtgatc atgtcctttg ggtgcctaga gctgcattat ccagcacagt 12000 agcaactagc cacatgtggg gctaccaagc acttgaaacg tggttagttt gaatttaggt 12060 gttctgtaag tataaaagat aaaccagttc aaagacagta tttcaaaaat aggatataaa 12120 gcctttttat attatgcatt gaaatattgg ggggtatatt gagttaaata aaatggtatt 12180 taatttaacg tattaaatat gtttcttttt acatttttta atggagctac tagaaaattg 12240 aaaattacat atgtatgtca cattatacac ctattggaca gcattagagt attcactctg 12300 attttctctt ggggcaggaa ttgccccttg tttcagttgc tattcccccg ttagcacccc 12360 gtgtatatca tgagtgcctt gaaggtttga caccacactt ttctgtttct tttattccct 12420 gctaatgcct accatatagt tgactagccg aaagttactg aataaacatt tttttttttt 12480 ttttttgaga tgaagtctca ctctgtcacc cagactggag tgcagtggtg cgatctcggc 12540 tcaccgcaac ctccgcctcc caggttcaaa cgattctcct gcctcagcct tctgagtagc 12600 tgggactaca ggcacccgcc accatgcctg gctaattttt ttatttttag tagagatggg 12660 gtttcaccat attggccagg ctggtctcga actcctgacc ttgtgatcct cctgcctcag 12720 cctcctaaag tgctgggatt acaggcgtga gctaccgcac ctggcctgaa tcaagatttt 12780 tttagccaca acttgtgcct tacaagtttt ttattctaag gattagacaa gtaaattatt 12840 tttcaccgta ttacattagt tcacctacta taccagtaac aattatttta ttcaaaagtg 12900 aaacagattt tactatattt atgggacaat aacagttccc ccttcctatt catattctta 12960 gatgctttct cataaattca gctgtgaaga gtcacattat ctttgatcca tgtgagacta 13020 tcttcttgtt ttatctactc ctgttagtct ccatatgtca ttactcttac cctccacccc 13080 acgttactgt tttaatgtgt ttagtatgtg tatgtgttct tacatagttt attttggtat 13140 gtttggtact ttatgtaaat attacatatg tcttatctgt gcgcttaata tattgcatct 13200 aactgctgaa tagcagtgag atgtatgcat ctaccacatt ttaccagtct gctcgccaga 13260 ggtgcacatg ttaattttct ccaactctgt caccacaaaa aaaatgcttc aacatcttca 13320 tgcatgtccc cttatggagc aatgtaagag tttctttggg atatatagct tggaatggac 13380 ttgatgggtc acaggaaatg tcagattgtt cttctagatg gctgcttcca tcaatagtgc 13440 accacagttt ttgtattccc gtttctctgc caacatttca gagctttctg ccttttccct 13500 atctaacagg cctaaggtga tactacattg tggttttaac ttgtatttct ctctgatttt 13560 tagaatctct ttatacattc cttggctttg ggtgcattct tgttttttgc gggggttggt 13620 gatttttatc aaatcaagga cagacatccc attctaatcc tagtttgcta taggaatttt 13680 gtgggggctt tttggtagag acagggtctt gctatgtttg cccaggcggg tctcaaactt 13740 ccgggtttaa gcaagcctct tgcctcagcc tctgaaggtg ctgggattac aagcatgagt 13800 caccatgcct ggccaagagt ttttaaatta taaattgttc aatcttatca aatccttttt 13860 ctatgttgaa tgaaataatt gcctagctgc tttcaaaagt gaaacgtgac tttgtgaata 13920 tttctaaagt acaaaatact tggaggacag ccatttattg tcacaacttg gctttgtagt 13980 ttgtttttgt ttttgttttt taacatttgg aaaccatacc tatgtgttaa gtaagtggct 14040 tgtgatttaa ggaatagaat gcatgtaagg gcacacttct tttgttttac agaagggatc 14100 aagttctgtt caggaagatt tgaaacatag tgtaaagttg ttgctgtttt aaacttgtaa 14160 acctgattct tctcatgtgg gaggtacgta agtaggcctc tcagatttca ggttggttct 14220 ctgcctggta gatacaaggg caaaactatt ctggaaccat attgatgtta aaaaaatttt 14280 ttacggactt cctgccggca aggatttttt taaaagattt tttaaatgtg tctaagaaat 14340 gttttaattt gtcttctgat tatacagtga tttacaattc atgcttcttt tatatgtggg 14400 caaaattaag aactaatatg taaaacaaaa tggagcacat tcccattttt cttattgctt 14460 tgagttgata tggcccctat gtcagtagtg ctttggctga acgtcacaaa gacctaacac 14520 agtgccctaa acaaataggg ggtttgtttt tctccaaaaa gaaatttggg gtagggtgcg 14580 gtggctcaca cctgtaatcc cagcactttg ggaggccaag gcgggtggat cacgaggtca 14640 ggagatcgag accacggtga aacctcgtct ctactaaaaa tacgaaaaaa attagccggg 14700 cacggtggcg ggtacctgta gtcccagcta ctcgggaggc tgaggccaga gaatggcgtg 14760 aacccgggag gcggagcttg cagtgaatcg agatggcgcc actgcactcc agcctggggc 14820 aacagagtga gactccatct caaaaaaaaa aaaagaaaaa aaaaaaaaag aaaggaattt 14880 ggaggtatgc agcgtctagc tttggatcag cagttcagta atattggagt tggtggtatt 14940 ttaaggtagt tttgtctttt ctgtcatgtt tgagaccctg tagctacagg ataatgtttt 15000 acagttacag ctacatggtt gcattcaagg aaggaggaag gaactgtact ggcaactctg 15060 tccttacccc cccaccctcc tttttttttt ttgagatgaa gtttctgtca ctcaggctgg 15120 agtgcagtgg cggaatctca gctcactgca actgtcgcct cccttgttca agcgattctc 15180 atgcctcagt cttctgagta gctgggatta caggcatgtg ccaccatgcc tggctaattt 15240 ttgtactttt agtagagatg gggtttcacc atgttggcca ggcaggtctc aaactcctga 15300 cctcaggtga tctgctagcc ttggcctccc aaagtgctgg gattacaggt gtgagccaca 15360 gtgcccggcc tcccctttta tgataaacaa aggcattacc aacccctacc ccctactaca 15420 ttgccaccta gtagacttta actaatgaga actggccaga actaagccag atggctatct 15480 ctagctgcag agaagtttgg gaaaacaagt atttggccaa tgtggtctcc ctaaacacgt 15540 gttcaacttc ttgaagaagg aaatctcatg tctttggctg tattcagcat ttttcagtag 15600 cagttacata ttctagtttt catggtggat gaagtggtac cagcttagtt ccacaggagg 15660 gagaagaatc aggactcatt taatccataa gaattaaact cagcagcttt tgagtaccca 15720 ttatgtgcat agtctgccat gaaatattat aaagtagtcc aggtatggtg gcttacacct 15780 gtaatatcag cactttggga gaataagaca ggaggattgc ttgaggccag agtttgagat 15840 cagcctgggc aacatagtga gatcctatct ctacaaaaaa ttaaaacatc agccatgtgt 15900 ggtgtacgca cctgtagtac cagctactaa ggaggctgag gtgggatgat cccttgagtc 15960 caggagttcg aggctgcagt gaggtatgat cgatcacact actgtactcc agcctgggca 16020 acagaacgag accctgtctc aaaaaaaaaa aaaaaaaaaa gttatcaaag aattatataa 16080 cattttgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtatgcag gttgtttatt 16140 ggctccttag gaagaaaaaa atacgtgtat gaaataattg attaacagta caagaactca 16200 taattatttg tttaaatgtt taatgttggt actgagtgct gtggcatagg aatagggaac 16260 tttttcttag ggtcggtgat ggattcttgg gaaaataaaa tgaagggcaa atggaagcaa 16320 aagacaattt aatttgcttc tgttttaagg gaacttttaa aaagttaatt cttaaatttt 16380 ttaatttaag gacttcgtca agaaacatcg aattaaatat attgagttta accctaacaa 16440 gcatcagatc tttcagtctg ctgtatgagg aaatacaagt ttctttctgc tttctattat 16500 gattgttgat ttgcatgagc gtgttttagg ttaggctgtg ttatgctgtg gtaatacata 16560 ccaccaacat cctcatgatt taacacaata aaaaacttct gctcacacca agtctgcttg 16620 agggctcatt ggctccgtgg aaactgatct ccatgtccag gccagtttgg tcttgtggcc 16680 gtccgtttta gggcagtgct tagggtcagg agagcaaaac acagggctgg agagttaagc 16740 acaggcaact aaatgcttca gcatgggaat ggcattcatt ttcactcaca ggatgtgttc 16800 agcttagtca catggctgtc tatgcctaac ctcaagggaa gggagtgcag tcctccagga 16860 actcagatgt aaaaaagcca gtatgatgga tatagtattg tctacaaaaa aaagtagcat 16920 aatagagtta gatacaagca aagtactcta ggatcccagt aaggagaagt aagaaattct 16980 gatgagatgc agggagagcc ttagaagggt tcatgaagat ggtgacagat gatacaaact 17040 cagtagaatg agcaggtttt tcttagattt gaagagaaga aaggacattc aaactaaggg 17100 aacaacactg actgagtcag agctagactc aaagctagag tctactggct gatgtcgtgg 17160 gagaggaaga agagatgagc gggccttgaa ttcgtttaac attcagcaaa aatgtattgc 17220 atgtctacca tgtgttaggc actgtttcag gtttggggaa tatctgataa tgaacaaaac 17280 acaaaaagcc tgtccttgtg aaggttacat gctagtggaa gaagcgctaa ggagttagta 17340 ttttattata agcacactgg tgagtaagca aagataggaa cattggatgc ctgtgaatct 17400 taagattttc ctggccgagc gcggtggctc atgcctgtaa tcccagcact ttgggaggct 17460 gaggtgggta gatcacttaa ggtcaggagt tcgagactag cctggccaac atagtgaaac 17520 cctgtctcta ctaaaaatac aaaaattagc cgggtatggt ggcgcgcacc tgagtcccag 17580 ctactcatga ggctgaggca ggagaattgc ttcaacctgg gaggcggagg ttgcagtgag 17640 ctgagattgc gccactgcac tccagctgac agagtgagac tccatctcaa aaaaaaaaag 17700 gatttttcta agatttctat tttacattaa atagaaggca gttggtttgc atgaaaatta 17760 ttatttcatc aatttgactg cctcattata gaaaacacat attgaagtca aataaaacgt 17820 agagatgaat ctctgaagtt aaaatatgtt atttgggacg caagaattgc aatttggggc 17880 acacagacca cagggtggtc ttcagtatgt atgaagaaca aagtaaagga tggaggtttt 17940 ataaagagaa gtgttacata ttttgaaaga caggtcattg gcactagtaa agttttgggg 18000 aggtggcaag ccctgattgg tgagtgacag tggtgggtaa tactggtctt agagtcagca 18060 acaagttgtt tcagtagcca ttagataaac tggtttcagg ttacaatagg cagtttcagc 18120 agccaggctt acagagaatt ataatcttgg agcaatgtta catccctccc cctcccctgg 18180 ccccccaccc catccctgcc cctcagctct gatttagttg agtacgaata ggatgaccca 18240 attagtatga tcagctttca cacatgaagg ttgaatttat gaaacatgta aatttgaaaa 18300 tacatgttta cattgcaaaa agctttttta acaaaaattt tggccagtag tataaattca 18360 gtgaatatgt tttcattgca cagatagtaa aaccacttga tacatacctc atggggatat 18420 ggactgcttt atgcgctgct ttttaccaca ccttaaatag tgcctggttg tgtgtgtgtg 18480 tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtaactc agtaaatatt caatgaatga 18540 atgaagaaat tcagaagata aaaaagagaa atttccctta tttttaacct tctgaacact 18600 atagtggtta gcatttatat catttattca gccattctaa tatttattga gtagcagttt 18660 tgtaccaact gctgttctaa gatataagga ataataaatt cctgttactg ttttcatgaa 18720 acttaaattc tagtgtaatt aaaatcatgt agaaataaac ataaacatga tcacccagct 18780 tcttttaaaa ctagactgca gttgatgtaa taatttgcat tacaacagca tgcatcttgt 18840 gcacaacaaa atctacctgt ttttgtatta aagccttagt gtcaaagctt tattatcatt 18900 ttagtctttt atcaactcca tatttataaa tggtctattt tcccaaagtt ctaatatgga 18960 aatcagaatc atttttgtta gaggaacaat gtttacaagt tgcctagatt tgcagtgagg 19020 tcataaaaag cctgtttaat ctaaaataga catttgcaat aaaaacagaa attgttgttg 19080 agatgccata ttatgtgata actgagtaaa acagacaaga gcctatcccc taacctggta 19140 tgtgaatgtg aaaatattag agaaataatg aagaagtaga tggagactaa tgaggaaatt 19200 ctgaaaagga catcttttga gaaattcaga ggtagatggc gcaagtttta aagcttactt 19260 tcactttaaa acacctgagt ttcatttcct ttgatttgag taaattacaa agcagtttat 19320 tagttaaatc aggcttgttt tgatttatag gtatatagag aaagacagga cattttcctg 19380 gacagaaagt tggaaaagag aaacgtgtaa agggggtgga aagtggctgt gagtgcatgt 19440 ggaatgagaa gcctcccttg ggcaggagtt aacagccaca atggggaaag gattgggaca 19500 tctaatcctt tgagtgtgtg tgggggccaa gggagcagta gtagcaggat gcctgcgcat 19560 acagccatgt acacagtgtg tacacagaag tgtaattctt gggcctgaat atttcatagg 19620 gtagagacat attgagtact ttattaagta agcatattga gtactttatt catctataca 19680 acaagggtcg tctattaagg gccacacagt ctagctgttt ctgtaaagca cattgttttg 19740 aattactgtt agcagtgtag tatagaagga aatcgggaat ccagattctc tatggatttc 19800 cccctgaaca gttatgtggc tgtatcctta tctagtgaat ggtgacatta gcttttcccc 19860 tgctgcacgg gctgtagtga ggctcaggtg tgcaggtgca cttactaaca tatggaaaag 19920 ttaagcactg tataaccaga aggtagccct tttactccta ttttatatga cagccacgta 19980 tgcaaaatat ctaatttctt cctgaacact caattgaact tcaacacttc gtatttttcg 20040 tctcagaatt tttattttcc tcaaatttat attatgtggt taaattcctt tgaagtgcta 20100 gatactttaa gattaaaatt aaagtaggaa tgttttttct tcaattagaa gctcttagaa 20160 cttggcaagt actctgtgtt ctctcatttc cttatctttg tgtatgctgt attgcagcac 20220 acgtcacatg gagatgggcg tcaagaagtt acctctcgta ccagccgctc aggagctcgg 20280 tgtagaaact ctatagcctc ctgtgcagat gaacaacctc acatcggaaa ctacagactg 20340 ttgaaaacaa tcggcaaggg gaattttgca aaagtaaaat tggcaagaca tatccttaca 20400 ggcagagagg taaataccag ttatgcttat ttctgttatg acagttgctc tgtttatttc 20460 catgtaagag aaagaaaaga atatagatat aggccttatt tctttttttt aagatggagt 20520 ctcgctctgt cacccaggct ggagtgcagt ggcatgatct cagctcactg caaactctgc 20580 ctcccgggtt cacaccattc tcctgcctca gcctcccgag tagctggcag tacaggtgcc 20640 cgccaccaca cccagctaat tttttgtaga gacagggttt caccgtgtta gccaggatgg 20700 tctcgatctc ctgaccttgt gatccgcccg tctcggcctc ccaaagtgct gggattacag 20760 gcgtgagcca tagcgcctgt aatatatagc tactatgtat tacatgtatt acatgtcaag 20820 ttctaaccac ataatataaa tttgtaatac atagctggga ttacaggcgc acaccaccac 20880 accacgctaa tttttttttt tttttgtatt tttgtatttt tgtagagacg gggtttcacc 20940 atgttggtca ggctggtctc gaactcctga cctcgtgatc cacctgcctt ggcctcccaa 21000 agtgctggga ttacaggcat gagccaccgt gcccaaccta ttttattttc aagacagggc 21060 cttgccctgt cacccgagct ggagtgcagt ggctcaatca tggctcacta tagcctcaac 21120 ctcctggggt caggcagttc tcccacctca gcctctcgag tagctgagac tacaggcatg 21180 cactgccaca cccggctaat gtttaaaaaa tttttttgta gagacagggt tctcaccgtg 21240 ttgcccaggc tggtcttgaa ctcctgtgtt caagcagtcc tcctgcctca acctcccaga 21300 gtgttgggat tacaggcatg agccaccatg cctcactaat taagcttttt cttttttggg 21360 gggttagggg ggtgtcgggg gttgggacgg agtcttgccc tgtagcccag gcctggagtg 21420 aagtggcatg gtctcggctc tctgcaacct ccgcctccca ggttcaagcg tttctcttgc 21480 ctcagcctcc tgagtagctg agattacagg cgcacaccac cacgcctggc taattatttt 21540 tttttttttt gtatttttag tagaggtggg gtttcaccat gttagtcagg ctggtttcaa 21600 actcctgacc tcaggtgatc tgcccgcctc agcctcccaa agtgctggga ttataggcat 21660 gagccaccac gcccagccta attaagcttt ctcaaaagaa catgaaacat atattaggtg 21720 tctgggagtt ttttgttttg tttttgtttt tgtttttttt ctgaggcaga gtctcactct 21780 gtcacccagg ctggagtgca gtggtgcaat ctcggctcac tgcaagctcc gccttctggg 21840 ttcaagccat tctcctgcct cagcctcctg agtagctggg attataggca tgagccacca 21900 cgcccagcct aattaagctt tctcaaaaga acatgaaaca tatattaggt gtttgggagt 21960 tttttgtttt gtttttgttt ttgttttttt tatgaggcag agtctcactc tgtcacccag 22020 gctggagtgc agtggtgcaa tctcggctca ctgcaagctc cgccttctgg gttcacgcca 22080 ttctcctgcc tcagcctccc gagtagctgg gactacaggc atccaccact gcacccggct 22140 aattttttgt atttttagta gagacggggt ttcactgtgt tagccaggat ggtctcgatc 22200 tcatctcgtg atccgcctgc ctcagcctcc cacagtgctg ggattacagg cgtgagccac 22260 cacgcctggc ccatctggga gttcttttgc tttcccatta ctttaaacgt tggaaatatc 22320 atagagtgtt taaatagtct ttacctttaa aaataggtaa tgttttgttc tttttcagta 22380 gcaaatgtct gctaccacag taaaaattgt ctttaacttc aggtatacac ttatgtgtat 22440 gagctcatgt tacttgctaa tcaatattat tgtcaatatt tacagattta tcttcaagaa 22500 gtttcttaat ctctctactt ttctcatagc gtatgtttaa tcggttattg tgtaggaagg 22560 cacatacttt cctaaccttt gtgaaatggc tttctgctca gccccgttcc tattaatcaa 22620 aaatacatgc attaaaacca caaaactaac tccctcctct tgttatactc atatggtaca 22680 gagcctgttt tgcctctttt tttttttttt tttttttttt tgagacggag tctagctctg 22740 tcgccaggct ggagtgcagt ggtgtgatct cagctccctg taacctccgc ctcctgggtt 22800 caagcaattc tcctgcctca gcctcctgag tagctgggac tataggcgtg cgccaccatg 22860 cccggctaat ttttgtattt ttagtagaga tggggtttca ccatgttggc caggatggtc 22920 tcaatctctt gacctggtga tctgcccgcc tcaacctccc aaaatgctgg gattgcaggt 22980 gtaagccagc gtgcccaacc tttgatgtag tttcttgcat ttaggtcttt gcgcctgctt 23040 ttccctgtac ctgaaatact ctttactctt ctgtttaggg aacatttctg agaagtaatc 23100 attgacaaat cccctttccc caggcatggg attccataaa aatcccgata ctccccttat 23160 atggcacaat ttacattgta ttggaattgc tcgtttgtct gtctccttct ctagtttgta 23220 ggctgtctcc agtctgtagg ggaagatgac agaacttaac acagtgcttg gaacctgtta 23280 ggtacttaat aaatatttgc taaatgaatg caataatact gttttttggc gggggggagg 23340 tggtgttggc aacagagtct cactgttgcc caggctggag tgcagtggcg tgatctcagc 23400 tcactgcaac ctcccgcctc ctgggttcaa gcgattctcc tgccttagcc tcccgagtag 23460 ctgggattac aggcacacgc caccgcaccc agcgaatttt ttgtattttt agtagagagg 23520 gggtttcacc atgttggcca ggctggtctc gaactcctga tgtcaggtga tccacccacc 23580 tcagcctccc agagtgctgg gattacaggc gtgagccacc atgcctggcc tgaatgtagt 23640 aatacttttg aaggaagctt ttaaaacaaa tgctctccct gtgcgagacc cttctgatag 23700 atgtgccaag actgtgacac atatggcgta gcattcatgc ctgcagccca ccaggcctgg 23760 ggctgcaaga gcatgagccc tgggtctgtc atctgtcagc atgcatcctc tgtttgcagc 23820 aatgtgccct gtaaatattt tcatttcctg tgtgtgcgga gacttggaaa aggttgggaa 23880 gcactggact gtgctaactg gaattttaaa agactgttgc cagaaatttg ccttttgaca 23940 attcattaat tttagtgata aaattcttgt ggatttttag ttaagactcc ttgttgctga 24000 gctgccttat agactgcttt tggaatctgt aaacagcttg tgtgggcgga gctgtccttg 24060 ttgtccttga ttaaagcttt cttgagataa ggaacggtga gacctctgag tttcttagtt 24120 cccaccagtt ccaggtggct acttgccagc ttcctcatgt gggcagagat ggtgggccaa 24180 gagagcgttg agacagacga acctcttcca tttaccttac ctgatttaca ttatctagag 24240 tctggcctgt ggtgtttact tctagtaggt tctttctcta gaaatgcttg aatatgacta 24300 gcagagtggt ttaatttaaa aactttctca gtttctcagt ttttttaaaa ctttgaacaa 24360 gttctcactc gtaagtggag ttgaacaatg agaacacatg gacacaggga ggggaacatc 24420 acacaccagg gcctgtcggg gtgaggggca aggggaggga tagcattagg agaaacacct 24480 aatgtagctg atgggtcgat gggcgcagca aaccaccatg gcacatgtat acctgtttaa 24540 cctgcacgtt ctgcacatgt atcccagaac ttaaagtaaa aaaaaaaaaa aaaaaaaaaa 24600 aaaaaaagga aaaactttga acaaaatcat tttactcttg tggggttttt tttgcctatt 24660 gtagatatga catataaaat gtaaacatta tagaaatata gaaattcatt atgtagaaag 24720 taagttcctt gaaatttgtc tcttcaaaga caatcacttt cagcagtttg gtgtatattc 24780 ccagagtttt ttatatagac attaactttt aaaaaatttt aacagaaagg gactcatgga 24840 acatgatgac agttttttta cttagtaatg taccttgacc atgtctgggt cttagtactc 24900 atagatctga catattagtc taaaggcagc atagtcttcc tggctatggt ggtaccatag 24960 tttatttcct atcagtgggc attagggttg tttctaaatt tttgttgtta caaacagatc 25020 tgcagtggac attctttagc attgcgtgtc cttgttaagt gtttctgtag gataattcct 25080 agaaatggaa gtcctgggtg aaagtggatg actatttgac attgtaatag atactactaa 25140 attggttttg aataaaaagt gatgtaccta ctttattatt ctaccaaaat ggaatagagt 25200 gttcattttc gtacatgctt ctacactgta gataattttt gtctatctga aggtctcaat 25260 ctgtttttgc tgctgtaaca caataccagg gactaggtaa tttataaaga taagaaattt 25320 atttcttaca attgtggagg ctgggaagtc caagatccag gggctgcatc tggccagggc 25380 cttcttgata catgaagggc gtgatggaag ggcaaagagt ggggaaggaa gggaagagga 25440 ggagaaagga agaaaagagg gcagagccct tgcaatctga ttgcctctcc agggccccat 25500 gccttagtac tgctgcaaca gcaattacgt ttcaccatga gtttgggagg ggacattcat 25560 tcaaaccata gcacgtatgg acaaaaaaag ggtgtagaga aaaccagaag ttgctttttc 25620 ccttttcact acttagattt atgatctttt tatatatttt tgtcattcat aatttctttt 25680 tgaggcaagt tccctggcca cccgcagtag ctcatgcctg tattcatagt gctttgggag 25740 gccaaggcaa ggaggatcac ttgagcccag gagtacaaga ctgcattgtg ctatgattgt 25800 accgctgtac tccagccggg gcaacagagg acactctcta aaataaataa aaataaaaca 25860 agtttcctgt ttatatcctt tattcatttt tctttttttt taatcttagt gatttaaacc 25920 ctttatgttt cagtcatctg tctgatgtgt ctgttaatgt aggttatgaa tatctttccc 25980 tagattactc cttttaactg tttatgatgt ctttcttgtt ttagggaagt ttttaagtat 26040 ttatgtggca aaatcattta atctttttct ttattacatt tgggtttcat gtcttcttga 26100 agatttgtcc cgtgacttaa aaaacagttc tataggccgg gtgcagtggc tcacgcctgt 26160 aatcccagca ctttgggagg ccgaggtggg tggatcacga ggtcaggaga tcgagaccat 26220 cctggataac acggtgaaac cctgtctcta ctaaaaatac aaaaaattag ctgggcatgg 26280 tggcgggcgc ctgtagcccc agctgttcag gaggctaagg cgggagaatg gcatgaaccc 26340 gggagacgga gcttgcagtg agctgagact gcatcactgc actgcagcct gggtgacaga 26400 gtgagactcc gtctcgaaaa aacaaacaaa caaacaaaaa accagttttg tattttcttc 26460 taatactcct cctatggctc acattttaac gtttaattca tgtggtatgt atttgggaag 26520 attagtatta ggtgtaggga tctgactttt tttttccact tggattacaa ttgtcccaga 26580 accatcagtt ctctcccaac aattgaagat acctgcgtta acacataata aattctcata 26640 tgtgtcctgt tatttctcca gtctgtttat ttagtcctat acccatactg tggtgttttt 26700 attactgcag ctgtggattg tatgttttag gtcaagttcc cctcctgatg attctttttt 26760 ttttctctct ctcttttttt ggtgagatgg agtctcgctc tgtcgctagg ctggagtgca 26820 gtggcgggat ctcggctaac tgcaacctcc acctcccggg tttaagcaat tcgcctgcct 26880 cagcctcctg agtaagtggg attacaggtg tgtgccacca cgcccagcta atttttgtat 26940 ttttagtaga gaggggattt catcatgttg gccaggatgg tctcgatctc ttgaacccat 27000 gaactgccca cctcagcctc ctaaagtgct gggattacag gtgtgagcca ccacgcccgg 27060 ccaatgactc ttatttttca aaattttcat agcttttttt tttttttggt catttactct 27120 tccacatgaa ttttagaatt aagttgccat tttctaaaaa cagagcatga aattttgatt 27180 agattagttt tcatgggatt agttggtttt ttgttttttg ttttttttac tagtagcagt 27240 agaatctttt caaatatgcc ttaatatagg cgctggggct tgcccgtggt gctcttgata 27300 tataaggccc ggacattctg ccagtttttt ctcgagcagt gacaccaaga agttaacagc 27360 caggtgaatg ttatgcacaa gctcatcgtc tgtcatcttc acgtggccaa cagccacagc 27420 caaacataac atgttcatat gaaacttgat tgtggacttc gcctcatcca ctttggccac 27480 cgtgttttca tcgtgtgtga gcagggaagg gaactttact gcctttgtta ggcctatgcc 27540 gaggattcgt gggatttgct ttatcagaga ctctgaagcc aaaaatgcat catacttctt 27600 ggccagcttc ttgaccagat tcttattctt gagttttttt ggcacctcag tgtccacttg 27660 gggggatatc cgtggccttg gtctcgtcac agtgctgctg ttcccccagg acccacactt 27720 gggacaggga gtggacttaa gcctgacggt gcccgagaag cgcttgtcct tctgggggtc 27780 atagttcttc aagctgatct gcaactccac catcttcagg aacttggggc actttcactg 27840 gttcccgtgc aggacttcct gcaccgcctc atccagggtg tcgtgagaga ctttgctgct 27900 tatgggttct catgccacgc taaccagaaa agagggttta gttggtttta taaattatta 27960 atagtttggg aggaaattga tattttaatg atgccaagtc tttctttctt ttttttcttt 28020 cttttttttt tttgagacgg aatttcgctc ttgttgccca ggctagagtg cagtggcgtg 28080 atctcagctc accacaacct ccgcctcctg ggttcaagca attctcccac ctcagcctcc 28140 cgagtagctg ggattacagg catgcaccac catgcccggc taatttttgt atttttagta 28200 gagacgggtt tctccatgtt ggtcagactg gtctcaaact cccaacctca ggtgatccac 28260 ccgcctcggc ctcccaaagt gctgggatta ccagtgtaag ccaccacatc cggctaatgc 28320 caaatgtttc tattcaggaa tgtgcatgtg tctcaaattt gttttcaggt tttgtaactt 28380 ttttcatcta tgttttatat aatgcttgtt aaattcattt caagctattt tataattttt 28440 gttgctaatg taaatggaac ttttttttcc tcaagaaggc aataatcata taacttcttg 28500 catttgggaa acttttgatc tctcatttgg aatgtcgaaa attaagttta tattgttttt 28560 taatagcatg cttttttttt ttttttgaga cagagtctgg ctctgtcgcc caggctggag 28620 tgcagcgcga tctcggctca ctgcaagctc caccttttgg actcaagcaa cctcccactt 28680 cagcctcccc agtagctggg accacaagca cacagtaccg cacctggctt attttatttt 28740 attttatttt atttatttat tttattttat tttatttttg gtagagatgg agtttcacca 28800 tattgcccag gctggtcttg aactcctgag cttaagcagt ccgcctgcct cggcctccca 28860 aagtgctgag attacaggcg tgagccacca cgcctggtca gttgtctttt tttgagaatg 28920 tcaaataaaa tgggatcata tggtatgtaa ccttttgaaa ctggcttctt ctcactcagc 28980 atcatgtctt tgagattgat tcaggctgtt gcatatattg acagtttgtt cctttttgtt 29040 gctgagaagt attctgttgt atggctgtgc tacagttggt attagctccc ccactgaagg 29100 acatttgaat agtttccagt gtttggcaat tatgaataga actcctgtac aggtttttct 29160 gtgaacataa gttttaattt ctctaggagt gacattactg gatcgtgtgg taaagaatta 29220 agtgaattgt taactagaaa actgatttta tataacagta taatattcct agtagaaaga 29280 aacctaaagc ctctggtctg gtctgtgaat tggtgtattt ttattactta gtacttttct 29340 tttctttctt tttttttttt tttaaagatg gacttttgct cttgttgccc aggctggagt 29400 gcaatggctc aatctcagct caccgcaacc tccacctccc aggttcaagc gattcccctg 29460 cctcagcctc cctagtagct gggattacag gcatgtgcca ccatgcccag ctaattttgt 29520 atttttagta aagacagagt ttctccatgt tggtcaggct ggtcttgaac tcccgacctc 29580 agatgatccg cccgccttgg cctccccagg tgctgggatt acaggcatga gccaccacac 29640 ccagctactt agtacttttc tcagctaaga tctttatttt tgtcagcgtt ctcttgccct 29700 gtgatgggaa cgtgatgtat cttgcctatt gaagaactaa aacctctgaa gaaaacccag 29760 agggttgtgg gaagagtggc attgatgatt tggggactta atttgttctg agtcagtata 29820 agttaaaggc ttattgtgta ttatgttgag agttttaact gctatccttg gggtaagaac 29880 agagatgtag aattatatcg tgaaaagaat tttctgaaag aagggagtag ggacaatcta 29940 tatcatgcat ttaagtcaca aggatgtcgt gaacataaaa gatactcacg aaggccgggc 30000 atggtggctc atgcctgtaa ttccagcact ttaggaggcc aaggtgggca gatcacaagg 30060 tcaagagatc gagacctgag gtcaggagtt tgggaccagc ctggccaaca tggcaaaatc 30120 ccatctctac taaaaataca aaaaatagcc ggggatggtg gtgtgcgcct gtagtcccag 30180 ctactcggga ggctgaggca ggagaatcac ttgaacccgg gaggcggagg ttgcagtgag 30240 ccaagatcat gccagtacac tccagcctgg caacagagta agaccttgtc tcaaaaaaaa 30300 aaaaaaaaaa aaaaaaagat actcatgaaa aactatatgt tcgttggaag aaagttgttt 30360 taatttaaca aacatgttgg aataagaatt ataaacaaga tacattaata tgtacactct 30420 gaagaccatt ttaaatcaag cctttaaata gcctagttat ggtgtaagtt cagtttagat 30480 gagacttccc acaggatgac catgaaccaa tcagaaatgg atatcaaaat gctcatttta 30540 tatattttta aaatacatgt accagttgtg aaattcatct gatttagctc ttaaatgaaa 30600 atgctaccca ttatccatat tgtcctggaa gcatctatcc catatttact atgaatctgg 30660 ggatgttagt tgcatctgct gtcttgcttt tgaccaactt ttcatagctt atggttattt 30720 tggtgtgaaa tattgcctgt tttacaccac agttttcttt caagttcatt attttccccc 30780 cactaatttt catagcctcc cagagacctt tcccctcaca cgaactacag aactcacttg 30840 tttcagtcat ttgtacttta ttggagttat ttattgttta aaaattattt tcctaaagta 30900 atgcatgcac atagcttaac aagtcaaata tactaaaaag ttctaatgac aatcagaagt 30960 ttcttttctt atccctcttt ttcctcagca ttattactca aaggccccaa cttttttaag 31020 ctctttcttc tgttactcca atagctttct ccaaatttat cattttaaac atcattgact 31080 tccacttccc acttccatat ctacatttct ttccctagct tttctattgc agtgatatat 31140 ttggcttatt tacttaaatt acgactgtgt aagtattgtt gagctttggc tatgatcatt 31200 tattttctta tataattgta aagctttgtc taaaattaat agttgtttct ttttctataa 31260 actcttaggt tttatgtgtt actgctaatt ggccaaaatg ttctagctta tctttcaaat 31320 attttgcagt actcacacat ataatataat gtattagctc aagagagaat cggtgagtat 31380 aaattagaga cagcaaggta gctctttcta ggcacagata agtgggatgg tagctacagg 31440 gagacattag gccaaggtat tttttagaag atggacaaaa tatcaacata tttctctact 31500 aatggggata atctcataaa aaggaggaaa ttggtgctgt cgaagagatt ttggaatagg 31560 tgaggggatg gtggaggcat tggtctttgg ataacatgga cagttcttcc atagaaatag 31620 gaggaaagca agccaggcgt ggtggatcac gcctgtaatc cctgcacttc gggaggccga 31680 agcgagtgga tcacctgata tcaggagttc gagaccagcc tggccaacat ggtgaaacct 31740 catctctact aaaaatacaa aaaattatct gggcatggtg gcgtgcccct gtaatcccag 31800 ctactcagga agctgaggca ggcgaattgc ttgaacccag gaggcagagg ttgtggtgag 31860 ccgagatcgc accactgcag tccagcctgg gcaacaagag caaaactctg tctcaaaaaa 31920 taaagaaaga aaataaagta aagaaatagg aggaaagcag agggtgccgg catcagtgca 31980 ggtgagggga tccttatggt agtaggagct tgtagaaatt ttctgagtgc aaactgtttt 32040 ttcagtaaaa tggaaagcaa actgatctgc tacgatgagg agatgtatta gagatgtgag 32100 gaggttaaaa taattgccag ataagcagga gagtgaatgg actagagaaa tgtaatagaa 32160 ttgccaggca gccttaaggc ttccctgagg ttggtgacca aggatttaaa gtgggaagag 32220 tcagcatggc gttgtgctac tctccagcca cattcagttg cccagctgca ggcatggcat 32280 ttgcagagat tggggttttg tcacattagt atagtgaaag gacaaaggag cattgttata 32340 caacacccca ggagcattgt tatacaacaa catcacaatg aaattagtga tataattagg 32400 aactaagaac ttgaggagga agactgaccc tatgaaggtg tggggtcagt gaattacaga 32460 tcctggtaag ttccaacagt tattagagta ctagattgag tgagcaggaa ggtagaagaa 32520 gttagttaaa gaatgggaag ctcaaacttg gatcatgaaa ggttttcaga tattgggaat 32580 gtcaaagact ggagtataac catgtaagta gcttaagtat gtaatatttt ttttctttct 32640 ggaagtttat ccttggtttt tagaaattag ccattgaggt gccaagggtt agtcttcatt 32700 cattgttctt ggtgctcatt ggaccctttt aatatagagg tttttgtctt tcagttctgg 32760 aaaattgtct cttttttaac ttaaaaatct tcattctgga atccttaagc agagactgga 32820 gtgcttgaaa agagcctcaa aatctatctt ctatctctat gtttttactt tctgggagat 32880 atttttaact tcatcttcca ttctttctgt taaaaatgtg tgtgtgggcc gggcacggtg 32940 actcatgcct gtaatctcag cacttgggga ggctaaggca gaagtgtcac ttgaggtcag 33000 gagtttgaga ccagcctggt caacatggtg aaaccctgcc tctactaaaa atccaaaaaa 33060 attagctggg cgtggtggcg cgcacctgta atcccagcta ctcaggaggc tgaggcagga 33120 aaatctcttg aacccgggag gcagaggttg cagtgagccg agatcgcgcc actgcactcc 33180 agcctgggtg acagagcaaa actctgtctc aaaagaaaga aagctgtgtg tgtgtctcct 33240 cttctctccc cagctcactc tttgatcatt ttcatctgtt tgttgttact gtctttacag 33300 catctagccc tggtattatg aatgttgttt cttctgctga ctccctgaga atagtaaagg 33360 cttttttttt tttttttttt ttttagctct tttataaagt agatttttgt tccctgcatt 33420 gcttctcttt ttttctgggg ttctttcttg ttaccgctct ttcatataga gactttctct 33480 ggatgtctgg tagcctctgg ttgtctgtac ctaatgtgga gccctaagca tctgatcaca 33540 agctgtgttt gtatgaatag gacatgtttg accacagagc tttcctattg ggagttgtct 33600 ggtgctttta ttgaagaccc cagatatttg tgtccttaga tttctgagag tatccagttt 33660 ctctagataa gaatcttctc ttctgcctgg gaggaactga tgatcccacc cctcagtcac 33720 ttagtcctcc cccgcctgcc ttttcatctg ttgcttcact cctgaaacca cgtttcttca 33780 tcctggggtg tgtgaaactt cctgagactt tgcataaatg cttatgttta gtcacgagtt 33840 tgttggtgaa gactatgtta acaactttac agaaagtttg gagaatagag aagaaaaacc 33900 ccacaatcct aatattctga ataccatttt tgtttttgta tattttccat atacatgttt 33960 tttcaacata tttttgatta taattttgta tcttgttttt tccgccctcg gaattttatc 34020 caaagcagtt ttctacgttg ccactaatcg tcataattgt tcatttcagt ggttgtgtac 34080 aatgccattg atgagaacag cagttctcaa acgtttctgt ctcagaactc ctttaaattg 34140 aaaattgagg ctgggcgcgg tggctcacgc ctgtaatccc agcactttgg gaggctgagg 34200 cgggcggatc acgaggtcag gagatcgaga ccatcctggc taacatggtg aaaccccgtc 34260 tctactaaaa atacgaaaac aaaattagct gggcgtggtg gcgggcgcct gtagtcccag 34320 ctaccaggag gctgaggtgg gagaatggcg tgaacccggg aggtggagct tgcagtgagc 34380 cgagattgta ccactgtact ccagcctggg tgacagagcg agaccccgtc tcataaataa 34440 ataaataaat agaaaattga gaactcccca aagcttttat ttattagggt tatatgtatt 34500 gatagctgct gaattggaag ttattactta ggaaacttaa aatatatatt aattcatcaa 34560 aaataataat cctgttacat ggtaatgtaa ataacatatt tttatgaaaa acaactattt 34620 tccaaagcaa attattgaaa agagtgaatt gtttaaattc tttgcaaacc cctttaatat 34680 ctggcttaat agaagttagc tgggttcctg tgtctgctct gcattcacac tatttaagta 34740 tgttgttttg gttgaagtgt ataaagaaat gtagctacaa aaggaaggga gcatattaat 34800 accttttcag gtgattgtgg atattgttct ttgacattat accaaaactc agcaagtggt 34860 ttcttggggg tgggtagaca gtgatgaatt tttattcaaa acaactgtag tgactgacaa 34920 aaaaagttaa aacccagaat gatttgcttc aattaaaaat taattatatc ccaagagcag 34980 gggtctacca ggttgtctga ggggaggggt gaactggccc agaaacggag caggtcaaaa 35040 ttcctctgct gatcagtggg atggggattg ggatcaagat tgcttctgtg aatagccact 35100 gcctggggaa catagcgaga tcctgtctct taagaaaaag aaacagaaac ttttggaaac 35160 aagcagacaa gggtgaaaga aagccttggt gtaagatata atttatatat tccatataag 35220 atatagaata catatgttat ataactaaaa agcataaaaa cagtcagcat gcttttagta 35280 caacctggag gtatggttaa tgtgcaggaa aaaagctcca tataagccta atccctatat 35340 ataaaaaatc acggaattcc acagttttga acattttttt ttctttttga gacagagtct 35400 tgctctgtcg cccagcctgg agtgcagtgg tgcaatcttg gctcactgca gctctctacc 35460 tcctgggccc aagcagtcct tccacctcag cctcctgagt agctgggacc agaggtgtgt 35520 actaccacat ccggctgatt ttttgtattt tctacagaga caaagcatcc ccatgttgct 35580 caggctggtc tcgaactcct gggctaaagt gatccttctg cctgggcctc ccaaagtgct 35640 gggattaaag ggatgagcca ccacacctgg ccaacacttt ttttgtgtgt gtggtaaaat 35700 acatacaaca taaaagttat aattttaacc attttttaag tgtgtagttc agtgacatca 35760 actatatgta cattattggg caaccatcac cactattcat ttccaggcag atagttaata 35820 ctcatttttt tgtggtttct atatttgcaa atttacctgt tcgaaaaaat ttatttgtaa 35880 cacccaaatc agtacagcgc tttggtaatc cttttcggac atatgcagaa cggtgaaaaa 35940 atatgcacat ccctagttga ggttcaacaa ggggacactt tgctttcttt ttttcagtct 36000 ttgtgccgta aaccagtgtc ttttttgtga tctatttaat gccttttttt tcatttttgt 36060 actttttgtt gatgattttg ccatttaaaa tgggccccaa gcatagtgct gaagtgctgt 36120 ctagtcttcc taacgcaccc ttgagtgaaa cttacctaac acctgcattt tctttgtaag 36180 gcacatcata gccttgccat ggataaaact ctggacagtt gagtttttcc ttgcactgca 36240 catggctatg gtgctttaca aagaagatgc acgtgttaga agtttcactc aagtgtgcgt 36300 tacagtgctg tggctgtgaa ttcagtgtta accaatcata tatagtaaag aaggtgttct 36360 taaacagaca cacacataaa acaaacttat gtattaatct gctgttgcaa aatgttgtga 36420 ccagaggctt gcagaaacct aacccccaga agcaatgatt tcgtattcac taattcagtg 36480 agcacagtaa ctgcatagaa tgaacaatga gaattgactg tagctccttt tttgaaagaa 36540 gaggcttaat tatggagcaa ggcatcagga aaaatcacag gctttcctca tttgtgtgat 36600 ttttttattt tgttcatgtc ttcaagagtg gagccctaag aagaaagctg agagctccca 36660 gtaaaggggt gacgagtgcc tggcttagcc tcatagaggc cagaggaacg tcggtcttta 36720 cactgggaca ttccacacac gcaacacaga gttattttct atggacagtt gagtttttcc 36780 ttttccaagc atattctagg gttgtctttc tgtgccctgc tacatgttcc actgtactgc 36840 tgtcagattt ttatctccca gaaattcttt gaagtcactt gtcttctggt ggcagccctt 36900 cccttctctt ggccattctt tgtcatttgg tctttgttct tcagtgggcc tatcaggtgg 36960 caggacagag aaatgcatgt gatcaatatg ccatctttaa ccaatgtcct cattatggtt 37020 atgaaaataa cttttcatgt aattattttt attcaagtta catattacac acagtttaaa 37080 gagtcaagac ttgttaaaaa agagtcccct agactcactg gtttccttgt ctcccaggat 37140 ggccactttt aatcttttaa agtgattctt tagtctttta aatgattctt tcatacttta 37200 taacatgctt gtagtcttac ttgtttttca ggtttaggca ttatctattg acattttatt 37260 taaagtcatc attgacttta aaaacacaaa aactctttac tgaaatgtaa cacaggttgg 37320 gtgcaatggc tcatgcctgt gaatcccagc actttgggaa gccgaggcag gcagatcacc 37380 tgaagtcagg ggttcaagac cagcctgacc aatatggtga aaccctgtct ctactaaaaa 37440 tacaaaaatt agctgggcat ggtgtgtgtg cctgtagtcc cagctacttg agaggctgag 37500 acaggagaat tgcttgaacc caggaggtgg aggttgcagt gagccaagat cgcgcgactg 37560 cactccagcc tgggcgatag agcaagactc catttcaaaa acaaaacaaa aagaaacgta 37620 acatacataa aaagtgccca aatcataagt gtacagcttg atgaattatc acaaagtaaa 37680 cacatttgaa taaccatgac ccgggcaaaa aatatactgt gacccacacc ctagaagcct 37740 ccctttggcc tccttcaaat cccttcacta ccccacctcc tcaagtgaac cactgtgcca 37800 ccttctaata ccataggttt tgtctgtttg ttgttttaac attgtactaa tagaatgtta 37860 gagtatttta ataatagtgt attattttgt gtctatttct tttactctac ttcatgcttg 37920 tgatattctt ccatgttgag gttttcattt gtaaccaact gttgtagttg tttgacttgg 37980 cctccatttg ctatggattg aatcgtatgc ctcaaaattc atatgtgtaa gccctaaccc 38040 ccagtgtgac tgtatttggt gatagggtct ttatagaagg ttaagtgaag tcgtagtatg 38100 aatcctgatt gatagggtta gtgttattac atgaagagac accagaacgc tagctttttc 38160 taccacatga gaacgcaggg agagggtggc catctgcaag ccaggaagaa ggtcccctcc 38220 agaactgtga gaaaataaat tactgttaag tcccccagtg tgtggtattt tggttatggc 38280 agtccaagat gactaatgca tcattcttca ggccactcta tccccctaga ggacttgcag 38340 tctttttttt ttttttgcct cctcttttac tgactgagaa tgttgttaac actttcagaa 38400 acagggtgtt aggatctttt gaccgttggt gaaccaacag aaagtggaag ataaggattt 38460 ttctccagtg ggtgtttgct gtggatttgt tgggagaagc ccatctcatg tatgagctgt 38520 cccatcccta ttttgtaata ttccactttg ctgctggcat cagagtgaat gtaaatggaa 38580 tttcccagaa atttccacag caagtggctt tctcacttag tgagccccct tggtgtttgt 38640 tgagtctccc tttacccaac cccacaatca ctggaataaa ggggtggtct tctctccagt 38700 ctacaccttt ctgaggcact gcaatcgggg ataccccagt taccacctct tttggtgacc 38760 ccaaggtttc agctttgcct gagctctcca ggctccagct ctgggccaag gtgattttgg 38820 tggagtcatc ccagcgggga gctaggagga agaggggatt aggaactccc gctcaggctt 38880 ccttgctgca cgtacacatg tcacatcaaa tgatggtgtt tcactctggg gacatagttg 38940 cttttgataa aatgactttt ctgcagtttg gttaagctga atgaaacata tttgagctcc 39000 tatttttgct tcagaggcta ttgaatagca gtacctgaag atgaaagtgt ataagaacag 39060 aatcagtccg ggcgcgatgg ctcatgcctg taattccagc actttgggag gccgaggcgg 39120 gtggatcacc tgaggtcagg agttcaagac cagcctggcc aacatggcaa aaccctgtct 39180 ctactaaaaa aatacaaaaa ttagctgggt gtggtggcac gtgcctgtaa tcccagctac 39240 ttgggggagg ccaaggcacg agaattgctc gaacccggga ggcggaggtt gcagtgagcc 39300 aagatcacgc cactgcactc cagcctgggt gacagagcga gaccctgtct caagagaaaa 39360 aaaaaacgca gaatcagaat ctttggaata gtgaccggat gccgtggctc atgcctgtaa 39420 tcctagcact tcgggaggct gaggtgggca gatcgcttga gcccaggagt ttgagaccag 39480 cctgggtaac ataaggagac ctcatttcta caaaaaatta gctggacatg gtggcacgtg 39540 cctatagttc catctactcg ggaggctgag gtgggatgat cacctgaacc tgcggaagtc 39600 gaggctgcaa tgaactgtga tcatgtcact gcactccacc ctggacaaca gaatgagacc 39660 tcgtctcaaa aaaaaaaaac aaaaaaaaga atctttagaa taggaggagg atattgagca 39720 gaaccataag aaactagtga ttaagagtcc aggtgtgaaa atagaaacca cgtctcagaa 39780 gctcagagag aagaagaact ggaaaggcag catctttgat ggatttggcc atgaagagga 39840 ggaggaataa agcagaatgg aggccagaaa agagtgtgta tgtctaacgg tttcattgga 39900 attgggagat ctaggtatac catgtaaaat tacttatgaa ggtaaagttt gaagaaaatt 39960 cattcgactt tgatttatat aatttttaaa acctcattat ctgttaaaac accagaagcc 40020 ctgttttttg gttactgatt atgagtgttc aaggcctcag atcaactcag catttattta 40080 tttatttatt tttatttttt tgagagggag tctcgctgtc ttgccaggct ggagtgcagt 40140 ggcatgatct cggctcactg caacctctac ctcccaaatt caagcaattc tcctgcctta 40200 gcctcccgag tagctgggat tacaggccca tgccaccatg cccagctaat tttttgtatt 40260 tttagtagag atggggtttc accatgttgg ccaggatggt ctttgtctcc tgacttcatg 40320 atctgcctgc cttggcctcc caaagtgctg agactacagg cgtgagccac cacacctggc 40380 ctatttattt attatttatt tatttattta tttatttatt tgatggagtc tcattttctc 40440 gcccaggctg gagttcagtg gcaccatctt ggctcactat aacctccgtt tccaggtttc 40500 aagtggttct cccgcctcag cctttcaagt agctggtggg attataggca tccgccacca 40560 agctcagcta atttttgtat ttttagtaga gacggggttt caccatgttg gccaggctgg 40620 tctcgaactc ctgacctcag gtgatccacc cacctcccaa agtgctggga ttacaggcat 40680 gagccacctc acccggacaa ctcagtattt attgagtact ccttatgtgc cagttagggc 40740 tataaagatg aataagatgt cttttcccca gttgagatat gcataagaca gttcgtgttt 40800 gcctgtttca gtagtcagta ttttcttttg cttattcagt ttccactgaa acttatgttt 40860 tccaaataca aaattagaat tccaactttg cgtttttcag gcgcacacac tcgttttccc 40920 tccactcctt ccccagttct gatttgcttc ctcctgaaaa tgggggttat agcccatcca 40980 ctccagggtc agaattgctg gataccttgt tgcagtgatt ttttctgtag ctcctctgga 41040 ggttatagac tcatagggaa taaataatgt ggtcaggtgg aggctttaga ccaagttaag 41100 cagggttttt aaaacatggt gttaatgtac ataatgcagc cattctcaaa agtatgacat 41160 ggggagaccc ctggaggttt tctgagaccc ttttaaggag tctgccgagt caaaactatt 41220 tttgtgatac tagtaaaaca tttgcttttt tcattctgtc tctcacaagt gtagagtgga 41280 attttccaga ggttacatga tgtgtgatat cacaacagat tgaatgtaga agcttgtatg 41340 aaaatttcat acaaatatat tatttgtgtt ataatgtgct tattgttatt ttgaaatcaa 41400 ttgttaaata cttttttttt tttttttttt tacagtttct cagtttttat ttgtattttg 41460 gtagatattc gttgatacaa cccacatttg ggggaatgag taatctttaa aagtgtaaaa 41520 gggaccaaaa agtgtgagaa tgtttgatgt agagaaactc caagctcatt ccctggactt 41580 tggctataga tgctttcttc aggggtgggt cttctgggcc tgtgtcctgc aggatgccca 41640 gtaggaagca cagcctcttt gatcttatgg gggtgggctc atttgctgtt ttggagctca 41700 ggtgcaaagc cgagttactt gatattccct ggtcatggca aagcctctct ccacttttct 41760 tcccttctat tgtcacttcc attctttttc catagtccat agaaatcatt tttacgtgac 41820 agtgatgagc tgaccagaag ctatagaagc tatagtactt cctatatcct aatgggtttt 41880 aaaaacactg gctggggcat actcaccctt tctgaacaaa ggctgctagg tgaccctgct 41940 gttgccgctc tgtatattca cttggttagc ccacagattg gtttggacct aattcccttt 42000 ccttctttaa ctgcccaaag ggcttttcta tttaaaaaaa aaaaaaaaaa atttttaaag 42060 ttttcagatt caatacccac ctatttgtat gcttacccat cccagagtca gctaaattta 42120 aagtgtcagt aaattgtaaa gaaaagattt gctacattgc atatgttttt agaagtgctt 42180 tctgtgtata atctctctct ctctctcttt ttttcttggt ttggaggaga ctttcccctt 42240 gtctataagt aacttaaata taaaatgtta taacaatacg tttattagtt tgaagcccca 42300 attttctttt ttttttttct tttaagtgaa aacaagttta taaaagaagt aaagaaacag 42360 gccaggcgct gtggctcaca cgtgtaatcc tagcacattg ggaggccgag gcaggtggat 42420 cacctggggt caggagttcg agaccagcct ggccaacatg gtgaaacccc atctctacta 42480 aaaatacaaa attagccagt cgtggtggtg cgcacctgta atcccagcta ctccagaggc 42540 tgagggagta gaaactatga aaacttggga gatggaggtt gcaatgagcc gagatcatgt 42600 cactgctctc cagcctaggc aacagaggga gactgtttca aaaaaaaaaa aaaaagaaac 42660 aaaagaatgg ctgctccata gacagagcag cagtatcagc tgcttgactg agtctactta 42720 tagttatttc ttgattatat gctaaacaag gggtgaatta ttcatgagct ttctgggaaa 42780 agggcagaga tttcctggaa ctgaaggtcc ctcccctttt aggggactat ttagggtaac 42840 ttcccaaggt tgccgtggca tttgtaaact gtcatggtgg tggtgggagt gtcttttagc 42900 atgctgatgc attataatta gcttataatg agcagtgagg acaaccagag gtcactttca 42960 tcgccatctt ggttttggtg ggttttggcc tgcttcttta ccacatcctg ttctatcagc 43020 agggtctttg tgacctgtat cttctgccaa gctcctccta tctcaccctg tgactaagaa 43080 tgcctgactt cctgggaatg cagcccagta ggtctcaggc ttattttacc cagccccttt 43140 tcaagatgga gttgctctgg ttcaaacact tctgacatat ttcccccctc ccttttacag 43200 ggggaccctt aatccttaag aattgtagcg ggacaaagat catctgtaac ttcttcaagc 43260 caaatagggg tgatgatatt cctgcctatt agggtctctt gtatttaggg tagggagaag 43320 tttagttaga aagcattgtt atagaagccc ttattttcag ttacacaatt ttataaagtt 43380 acaattgctt attgtaacca gctgagtttt aggttttgtg gtttgttgct tgcttgcttg 43440 cttgcttctt aatgccatat atcttggcat ttatcagtcc ataattacta aattcttaaa 43500 atccataaat atttattatt ctttctaggt tgcaataaaa ataattgaca aaactcagtt 43560 gaatccaaca agtctacaaa aggtaagatt ggttcaatgt ctagtacttt ttaaaaaatt 43620 atcggtgcta attgccatct aatttgtccc ttaaataccc caactgttat tttatgttta 43680 atgccataaa gcttcctatt cctcaaatga atgcaactta atgtagtatt tcatgaaaaa 43740 ttgttgggtt atctttggtc ggagattatt tttaaatgtt cttaacagca caaactaaag 43800 gctgtgcttt ttttttgtac ttttttattg atatagttgt acgaggctgt actttttatt 43860 gaatattctt aatattgact agagtttttt taaaaataat acagtacaaa aatattttca 43920 tgtaattgat attgtgcaaa tttgtacctt atagctaata ctgaaaattt taaagtgaac 43980 actttggctt ccttaaactc ttgacacacc acatctgatt aaggactgat gattaatata 44040 aaagggaaat gttaaagtaa aactgaaagt ggaaaagcat gttcataaag cagtattgcc 44100 attagtatca tttaacatag atcaagtctt tgccctagtg ccttttagaa tcacctgggc 44160 agatttttga aagtcttaac acttacataa taatgcctaa gcaattaggt tgggcccagg 44220 cataggtatt tcagtggtag gacatcagca aataattaat gatcttcata ataaaatcct 44280 tctagttgtt tttagcttcg cagagtcttt ttacatactt catttgattt ctagatttga 44340 cctgtagtag ttgagggggg tacagagacc atgaagtagg aacaggaagt actgaatttc 44400 agtcctggct ctgccactta gccgaatatc tgaccttgag caaggcactt aactttgctg 44460 gaccttgggt ttctcatctt taaaatggag ataatttcat cttcttatga tggatgtgag 44520 tattaatgaa ataatgcata taaaagggct ttgtaaactg cacagcactg cacagttgtg 44580 cgatattttg ataagagcag tgctctatat acatttctaa tgtattcctc atggttattt 44640 atatcctggt aattttgaag ccctgtccct gttttgtgtt tgggaggtgg ggagggagat 44700 agactttctc tgcactaaaa atgttatttc aacaagtggt tctttgtgat ctgcaaccca 44760 taatgggcct taaggcttct caaggtgggc atgaaacatc taaaaattcc agtcctttgt 44820 cctgaagagt aaattttatc tatttcctta aacttccata tcttcagcta tctaaggatt 44880 tggtactaat aaatacatta gagtttaata caatgtggta ttcagaacta aaattaacat 44940 acaaggatgt ttaatgagga ctctaattga gtattctatg atgttacata ccttccacta 45000 ttcatgtgta cctggatata attgtaatcc taacagtttt cccggaaaaa tttttaccat 45060 ggtgttgtat caagttggct gcagtttttc cttcttgttt gcttgcttcc tttagtcatt 45120 ttttatttta ttttattttt ttggtaatga tttttcattg ctgctcttcg tttattattt 45180 gtcacatcat tatcagttta gtgcatggta gtttggttag cccattagtg tgattatgca 45240 tatttgcatc tgaattaatt ttgacttata ataataatta aaactttaag atttgttttt 45300 ggtattattc aagtatgaac cattcactga agacttattc taaagccagc tctatcccaa 45360 gttaaataac ttgataagta taatattttc agtttatagg ccattgttta aaattgtgta 45420 tataacttca tatgcttatg aagtaaatta gaagcatttt acatacattt gtttccaatt 45480 ctttttcacc aagtatctta tccccagccc ccccatcttc ccccaccccc agcaattttg 45540 caacaatgta gaatgtacca ggcaactttg tcagccccct ttgcagtcat tcttgcatcc 45600 ttttttcttc tcagcaccat tcatagttgt atcacacttg cttcaggaac agctctctta 45660 gatagtgatt taaagctgtg gagcatgccc tagcagctaa cattcatcat aaggaacatt 45720 ctgccatcag cagtgtgcaa tctctttttt accaccacta ccgaaaaagg tggactggct 45780 cattcaggca gtattttaac agtgaacaca cgtgttaaaa tatcggtttc atgatattca 45840 ggcttgcata ctgggtcatg aacatttact aaatgcacat ataagtaaac ctgagttgac 45900 tatgtttgca aattgatatt attttcacag atgagtctct tcatttttcc tttgtatttt 45960 tgcagccatt gaattaataa acgttagatt ctggtgagag atttattttt ctccattgtt 46020 ttttaagaga tgggtcttgc tctgttgtcc aggctggact cgaactcctg ggcttaagca 46080 gtccttccac tttagcctcc caagtagctg ggactacaaa tgcacaccac agcatctggc 46140 tatatacctt ccaatgtctc actagtatat ctggaatact attctattct tacgcatctt 46200 ttaggtcaat tttttgtttg ttcgtttgtt tttgtttttg tttttttgag atggagtctc 46260 gctctgttgc ccaggctgga gtgcagtagt gggatctgag ttcactgcag tctccctctg 46320 cctcttgggt tcaagcaatt cttctccctc agcctcccaa gtggctagga ctacaggtgt 46380 acgccaccac acctggctaa tgtttgtatt tttagtacag atgggggatt tgccatgtta 46440 gccaggctgg tctggtctca aactcctgac ctcaggtgat ccacctgcct cagcctccac 46500 ttgctgggat tacacgtgtg agccaccatg cccaggccaa ggtgtgtgga tcacttgagg 46560 tcaggagttc gagaccagcc tggtcaacat ggtgaaaccg cgtctctact aaaaatacaa 46620 aaattagctg ggcgtggtgg cacatgctta taatcccagc ttctcaggag gctgaggcag 46680 gagaattgct tgaacccagg aggcgacaga acaagacttt gtctcaaaaa aaaacaagaa 46740 atttgtaaca tgtaatgaaa taattgattt tttgtaatgt atttatgata gctggttagt 46800 tagccaacct tgagcacatt cagggatgga gacacactcc ttcccgtagc atcttgttcc 46860 agtgttcagt ggctttgcta ttagagcctt ttccccttac ttcagactag catctgtctt 46920 ctagaaactt ctacaacact gtttaccatt tgtcatatgg agttatagtt actccaaaca 46980 taaccagccc ttagaagttg ctattttggg tcttgttgat tttctcttat gcagagtaaa 47040 tcccctcagc ctccttgggt actcccacag gagtttcgct gaacagaagc ctactttatt 47100 ccatacttgg aattgtttcc cccctcccac ctgaatttgt ttgattcctg ctaaaattca 47160 tttaattgat tttctgcctt aattccagtc ttttggggga ttttggaata atgggtcatt 47220 tgtattagct gctatgtaaa ttagatatgg atgtctccat ccaattaaaa tattaaacaa 47280 gacaagaaca agacttttca atggagaatt catccaagtt gatactgctg acccagtagt 47340 aaagcatttt ttgagtaaga tttaaccacc attcttaaat ctagactgag taggacagaa 47400 agggaaatcc ctgtgttttt aaattttaat gagatttaca gaggaagaaa aaaaggaaca 47460 ttaaacacaa atatatgcaa aatacccagt aggatttatt aaagcattat tgactatgga 47520 tttcatcagg gtctttgtgg ggaaatcctg cttataccca cctcaatctt cctccctccc 47580 ttccttccac tggccttatc gtgattgtaa aatagggcaa cactacattc cataaaatag 47640 aataagtgtc cctatgagct gagcagaagt tggctttaga gacaggcaaa ggctgaagaa 47700 ggctgaggca ggcccggcac agtggctcac tcctgtaatc ccagcacttg gggagcctga 47760 ggcgagagga tcactcgagc tcaggaattc gagatcagcc taggcagcac agagagaccc 47820 gtctctacca aaaaaaaaat taaataaatt aaccaggtgt ggtggtgtgt acctgtagtt 47880 ccagctactt aggaggctaa gatgggagga tcacttgagc ctagaagttg gaggctgcag 47940 tgagctgtga tggtgcccct gcattccagc ctgggcaaca gagcaagacc ctgtctcaaa 48000 aaaaagctga agcaaagaac aaagagtgta ttggtccttt cagaattcct tttttttgta 48060 aggcagggac agggaaacag aacaatagac cacaatagac tataccacat agaaaagtaa 48120 ccgattagtt aatgtcaggt tacttcacac tacatttttt tctgtaagga ttaaagcagt 48180 aggaactttg ttattgtgcc aattgaagtt ttttcttttc ttttcttttc ttttcttttt 48240 tttttgagac agagtcttgc cctgtgaccc aggctggagt gcagtggtgt gattttggct 48300 cactgcaacc tctgcctccc gggttcaaga gatacttctg cctcagcccc ctgagtagct 48360 gggactacag gtgtgtgcca ccacgcctgg ctaatttttg tatttttagt agagacaggg 48420 tttcaccatg ttggtcagac tggtctccca actcctggcc tcaggtgatc tgcctgcctc 48480 agcctcccaa agtgctggga ttactggcct gagccaccgt gctcagccag gacagtttaa 48540 atcttcaatc aggggctgga cgcagtggca cacgccaata atggcagtac tttgggaggc 48600 cgaggcgggt ggatcacctg aggccagggg ttcgagacca gcctagccag catggtgaaa 48660 ctgcatctct actaaaaata caaaaattag ttgggcgtgg tgacataccc cagctaccca 48720 gtaggctgag gcacaagaat cgcttgaacc tcggaggcgg aggttgcagt gagccgagat 48780 cacaccactg cactccagcc tgggtgacgg tgagactctg cctcaaaaaa taaataaata 48840 ataaactgtc ctgtttggga aattagctgt tatctccttt tcttctgatt tctaggaagg 48900 tcagataaca acttaattta ggtttgggaa tgtggaatct tagcagggat tactccattt 48960 ttaattttaa cctggtttcc tggggcctaa tgcaggagtt tagtcccaaa caatggcttc 49020 ctataatttt tatttaacaa ttcttccctt ttggtcaggc tttcacctag gtaagagtgt 49080 aaccaaaacc taggatatcc ctgctgttct cagttgccat cattttgagt ttcccgtttc 49140 agcatgccgt tgacagggtg ttctcattat cgtgtgtttc ttttgagttt ttgttgttct 49200 agccagagag aaccatttga catctgatag gtggctacat ggagacactt aacactctgg 49260 gaggatacag cgtaccagag agactaccat tatgactatc atgaggatca caccaagcat 49320 ttagagtatg ctcctaagcc agggttttta tgaaccaaat caactaaaat tgtatagcca 49380 aacaaggagt ctgccaattt taaccaagtt gtccgctcta ccatacccaa atcgcaacga 49440 gaagtgtcat gaactgcaca gattcctcca tgtttagtag gcatcccagc attccgtgac 49500 cgggctaagt ataaagcagg gagtgcaagt tacccacaga agctactgac tgtgaaattt 49560 tagctacagc atgatcctgc caaactgaaa gaggtaggca ttagtaagga aaaattaaga 49620 ggggcaaaag cattgttatg aagtcttgtt attatgacag tcttgggaaa agctgtccat 49680 agcatgcagt ccacaacttc tcgtcctggt tcgcagtttg aatgtctctg gttgtggcat 49740 ctggcattct ggtgaattct ctgtgtggcc tacacatcag gcatgagact cgaaatttac 49800 atcaagctgt cagctttggt ttatagggct tctgaaattg agcagctcat tcttcattgg 49860 caagttgtag ccagatatta aagaaaacta gaagaattca gaatctagtt cagtcaggaa 49920 taatcttatc aggtcatttc tggaagtcaa ttctgtgaat taatatttta gcagcgtttt 49980 ttgcttctaa tttcaaattg gaaggtatag ggttattcat tgtgcccctc aagcctggtc 50040 cactccgata cccctcaagc accagacacc tacccaggaa gagggaggct gttttctttt 50100 taaagctagt atttgctttg gagccactgt tagtgcttag agctctgtag cagtagcagt 50160 agtggccttt tttggcgtcc tagggaatct tggtcatggg atgtagcagg gaaacttata 50220 atttgaaagc aaggagaacc agttcatgct tttcatgtgg tctgtcgtgt aatgactttt 50280 actgctgcca ccactgggtt attttgggca ctgttgattt agttttctta agccagtact 50340 tttcaaccct tcttgtgcat tagaaacatt tgtggagttt gaaaaaagca tatttccttc 50400 acagtacttt cctccctaga ggtcctatct tggcgggtct agaaatgtgg tgatctcagt 50460 acacacccca gatgagaatg gctcctagtc tgtcttcact tcccccaaag ttcttaattt 50520 ctctcttctt ccaattttac ttatttttat ttatttattt atttttttga gacggagtct 50580 cgctctgtca cccaggctgg agtgcagtgg tgcgatctca gctcactgca agctctgcct 50640 cctgggttca cgccattctc ctgcctcagc ctcccgagta gctgggacta caagcacccg 50700 ccaccacgcc cggctaattt tttgtatttt tagtagagat ggggtttcac cgtgttagcc 50760 aggatggtct caatctcctg acctcatgat ctgcccgcct tggcctccca aagtgctggg 50820 attacaggcg tgagccacca cgcccggcct atttattttt tattgattga gtgattgatt 50880 gatggggtct tgtcctgttg cccaggatgg aaggtagcag caagaccaga gctcactgca 50940 gcctggaatt cctgggctca agtatctttc cacctcagcc tcctgaggta gctgggacta 51000 taggcgcgca ccaccacacc caactaaagt aacctatttt atttatttat gagacggagt 51060 ctccctctgt tggccagcct ggagtgcagt ggtgccatct cggctcactg caacccctgc 51120 ctcccgagtt caagtgattc tcctgcctca gcctcccaag tagctgggat tacaggcacc 51180 cgctgccaca cccagctaat ttttgtattt ttagtagaga cagggtttca ccgtgttagc 51240 caggctggtc tcgaactcct gacctcaagt gatccacccg cttcagcctc ccaaagtgct 51300 gaattacagg tgtggccacc gtgcccggca aagtaaccta ttttaaagat gaccccttcc 51360 ccaaatattc taagtttcat agacagctgc tactgccact gccactgctg aatattttct 51420 ccctctctgg aactcttcct aggaatgcct tagtacccag ttcctggcct cctttctcta 51480 ttgggtgaag cacaagttat tttggaaatc cttttcattg aacaactctt ttcatctttt 51540 tcttttcttt cttttttttt tttttccaac tatctttctc tttcattttc ttatattgac 51600 ttttcaggcc attaacaaaa gtagtaacca ctgtaaaaat agttgttaca gtcttgtctg 51660 ttatttaaaa tccagtgtct gcattcacat cagtgtcata aatctgataa attaatacaa 51720 ataggagatg gcaaatgaaa tccaagacag gcacttctta tattgattga ttgaggctgg 51780 gtcttacttt gtagcccaag ctgaagtgca gtgtcatgat catagctcac tgtagcctgg 51840 aacttctggg cttaagcaat cctcccgtct tggcctccca aagccttggg attataggca 51900 tgagtcacca cccccagcct gattatacat atttaaaata ttgagaatag actgggcacg 51960 gtggctcact cctgtaatcc cagcactttg ggaggcccag ctgggtggga tcacctgagg 52020 tctggaattc gagactagcc tgacgaacat ggtgaaaccc cgtctctact aaaaatacaa 52080 aaaaattagc caggtgtggt ggcgcatgcc tgtaatccca gctacttggg aggctaaggc 52140 aggagaatca cttgaaccca ggagacggag gttgcagtga gccaagatgg caccactgca 52200 ctccagcctg ggcaacaaat gagaaactct gccaaaaaaa aaaaaaattg agaataaaac 52260 tgtatatatg tttgtttatt tttagatttc aaaagagaag aaatagaaaa accggctttc 52320 agtattgttg cttctttatt ccttataaac ttttaaattt cttgccagac ttatttttgt 52380 tttggcaata aataatacgg gttgatattt agaatacatt ataaactcgg gaagctgagg 52440 caggaaaatg gtgtgaacct gggaggcgga gcttgcagtg agccgagatc acgccactgc 52500 actccatcct gggcgacaga gtgagactcc gtctcaaaaa aaaaaaatac attataaact 52560 gagaatagta gagtgtattt tagagattga ttgtttgttt ttagaattga ggtctcacta 52620 tcttgcccaa gctggtctca gactcctggg ttcaagcatc ctccttccga ggattccaat 52680 ctgccttcca aggagctggg attacaggca cacaccacca tacccagcta aagtatattt 52740 ttcattgtac caaggactta ttatgctatt ttagaaaagt caccaaggaa ccaagtattt 52800 taagtgggtt aaacttagag catagcctgg acccatttgc agaaaatata aacttgggtg 52860 caaattaggc ctttgtgaga gaattataac agtaacatca gccagaatca caaacacatg 52920 tcaggagttt attgtgtgcc aggtgctgtt ccatgcactc tgtatgtcaa gccatccttt 52980 cattgtagag gtgacagggt cagggaggtc aagttaccta gacttagtga gtggtacagc 53040 tgggttggga ttaggtagta tagctcctgg aatcaaattc tacacacact ttatcttata 53100 gcattgcatg tacttttcac aacagtcctg tgaagcagtt gttttctttt tttttctttg 53160 agacagagtt ttgctcttgt tgcccaggct ggagtgcaat ggcgtgatct cggctaaccg 53220 caacctccac ctccctggtt caagtgattc ccctccctca gcctcccgag tagctgggat 53280 tacaggcacc cgccaccacg cccagctaat ttttttgtat ttttagtaga gacagggttt 53340 ctccatgttg gtcaggctgg tctggaactc ccgacctcag gtgatccacc tgcctcagct 53400 tcccaaagtg ctgggattac aggcgtgagc caccacgcca ggccgaagta gctgttttca 53460 aaatctatct tataggtaag taggttaaag ctctcaagat tttgtggttt gttcactcat 53520 ccagtgaata agggactgaa ctagccttag aacctaaatt tataacatca aatagctttc 53580 tttgcataag ttcccttgga gccagtcact catagtactt tccatatggg aaggataaca 53640 aaggaacata tggaaattca gcttgcatgt ggtagatacc tagtaaaatc tgataagtta 53700 attttgttag taccaaattc attttaacac taaattaatt tttcctgatg ttgctctgat 53760 tctaggaatc ttgaccacca gaaccttgag tttgggagat gggaagattc ttagtttgtc 53820 tttgaaatat ttgatcataa aaaacacatt attggccagg catggtggct caagcctgta 53880 atcccagcac tttgggtgac caaggcaggc agattgctga gctcaggagt tcaagaccag 53940 cctgggcaac atggcaaaac cctgtctcta catacagaaa ttagccagat gtggtggtgc 54000 gcacctgtag tcctagctac ttagggggct gaggctggag gattgcttga gcccgggagg 54060 tcgaggctgc agtgaaccat gttcatgcca ctgcattcca gcttgggtga cagagcaaga 54120 ctccttctct aattaaaaaa aaaaaaaagc acacacattg ttgctactct tattttacat 54180 ttttagaaaa ctgatttgtt gtattactca gctaaagcta agcaattttt taaaatttag 54240 tttttaattt ttattaaata ctgactgcat acaaaagaat gttccatgtt tagcatataa 54300 atatgtatat gagatgagca cccttatact acctgcttca agaaatagat cttcaaccat 54360 acctttaaac cagtcccata ttgtgcccct cctcaacctg attccccttc ttcattgccc 54420 acctactggg taaacactaa caaggatttt gtgttattat tttcttgctt tgtccgtgta 54480 attttaccac atatggatca ctcatcaatg tagtgtaaag gttttttttg gtctgttttt 54540 tgtttctttt gtgtttgttt gtttgtttgt gactgagtct cgcactgtct cccaggctgg 54600 agtgcagtag tgcgatctca gctcactgta acctctgcct cccaagttca agcgattctt 54660 gtgcctcagc ctcctgagta gcagggatta caggcgtgcg ccaccacacc cagctaattt 54720 ttgtattttt agtagagacc aggtttcacc atgttggcca ggccggtctt gaactcctga 54780 cctcaagtga tccacccacc tcagcctccc aaagtgctgg aattacaggc gtgagccacc 54840 gcaccccacc aatgtagtgt aaagtttaaa aaattgtata aatggactca tgctatgtgt 54900 attctcctat tggcttttgc tttgcttcct tttttttgtt tttttgagac ggagtttcgc 54960 tcttgttacc caggctggag tacagtggcg tgatctcagc actgcagcct ctgcctcctg 55020 gattcaagtg attctcctgc ctcagcttcc aaagtatctg ggattacagg tgtgcaccac 55080 cacacccagc taatctttgt gtttttagta gagacgggtt ttcaccacgt tggccaggct 55140 ggtctcgaac tcctgacttc aagtgatcca cccacctcag cttcccagag tgctgggatt 55200 acaggtgtga accaccatgc ccagccgctt ttgctttctt ttacctacat tgtgttccat 55260 gttgatgtat tttgctgtaa ttcaccttca ctgctatgtc cattttcaag aatatgtagt 55320 atcccattat gtgaatatgc cactctgtat ttattctgtg attcagagac atttggattg 55380 ctttttgtat tgcaaacatg actgctgtga attgtcctct gtgtgtcctg gcacacttgt 55440 gaagagtttc ttaaatatat atacttagaa ttcttgtatc acagagtctg tctgcacatc 55500 ctccttcttc attagagaag gccaaattat tttcagagca ggtatgctaa tttatatttc 55560 catcagtagt gtgtaagaga gcacaagtaa tgtagcatcc tcatcaaaat ttgattttct 55620 ctgatttttt aaaatttgtg ccatcacagg agaattgctt gaacccggga ggtggaggtt 55680 gcagtgagct gagattgcgc cactgcactc cagcctggcg acagagcaag actgcatctc 55740 aaaaaaacaa aaaaattgtg ctatcaattt ggtaaatata aaagtgatat ttggccaggc 55800 atagtggctt cctccttaaa ttccagcact ttgggagcct gagatgggaa gatctctcca 55860 agccaggagt tcaagaacca gcctgggcaa cagagcaaga ctctgtatct acaaaaagat 55920 tttttttttt tttaattatc tgggcatggt ggcacatagc tgtggtctca gataattggg 55980 aggctgagga ttgattaagc ccaggagttt acagctgcgg tgagctgtga ttgcaccact 56040 gcactccagc cagggtaaca gagcaggagc ccctcttaaa aaaaaaaaaa gaaaagaaaa 56100 gaaaaatctc attatggttt taatgtaaca ttttcctaat tactgatgag gtcatgcatt 56160 ttttcatatt taggtttttt gtttcttctg tgaagtacgt attcatcttt tgtttaattt 56220 ctagtggatt ttttcttagt tattttagga tatcattata tatcatggat actattatat 56280 atgtggcaca caccttctac ttgatggtat atcttcactc tcttgatact gtctctcagt 56340 gaatagcaga ggctcctaat tttaaagcag ttacatttat cagtcttttt ttaatatgct 56400 ttgcactttt tatgtctttt ttgttttttt gtttttttgt ttttgagacg gagtttcgct 56460 ctgtcaccca ggctggggtg cagtggcacg atctcggctc actgcaagct tcgcctcccg 56520 ggttcacgcc attctcctgc ctcagcctcc cgagtagctg ggactacagg tgcctgccat 56580 cacgcctggc taattttttt tgtattttta gtagagacgg ggtttcaccg tgttatccag 56640 gatggtctcg atctcctgac ctcgtgatcc acccatcttg gcttcccaaa gtgctggaat 56700 tacaggtgtg agcccccacg tccggccttt tttgtgtctt ctttaagaaa ttcttctcta 56760 cctggggtca taaacatatt tttctctgtt gtcttcacat gtttaaagta atctttaagc 56820 caccagcagt catgtgttac gaggtaggga tccagtttca tggtttttcg tatgtataat 56880 cacttgctgc ctcacctctc ctttctttgc tgatctgcat cgtggacaga gtctccgtgt 56940 ctaaacacac actttgttct tcagaagtgg ttattctggt tcacttaaca attgccattt 57000 gaaaagcaat tatttttatt ttattttcat atttttaaat tttattttta atgataggac 57060 accaaatgat aaccagaacc ccaaaataca taggtgaaaa tctgagctaa attaatagaa 57120 acacaggcat gacagagaag actttatcac ttaactctct taggccattc attaattaca 57180 cagtaagtag ggggaaaagg tacatatctt aatatacagc acctccatgg ccaggtgcgg 57240 tggctcacac ctgtaatccc agcactttgg gaggctgagg tgggcggatc atgaggtcag 57300 gagattgaga ccatcctggt gaacatggtg aagccccgtc tctactaaaa aatacaaaaa 57360 aattagctgg gcgtggtggc gggtgcctgt agtcctagct actggggagg ctgaggcagg 57420 agaatggtgt gaacccgggg ggcggagctt gcagtgagcg gacatcgtgc cactgcagtt 57480 cagcctgggt gacagagtga gactccgtct caaaaaaaaa aaaataacag cacttccatt 57540 agcccatagt atagctgttt taatcccctt ttcaggatac ataacagctg tgtgctagga 57600 tattatcgtc ataactattc caatgtgtga tgactatgag gtatattatt ttctcagagt 57660 tgggcaatac ataacatttg aataagccag actaacatct ttctaggcta tagttctaat 57720 gtagagcaat aaaaatagaa ccaaacaaaa tatacaaaca tgtggaacct aaaacacaca 57780 gaattcttgg atcaaagaaa aattgcaaac aaaaattaca gtatatctag gaattaatga 57840 taataagctg tgtgtcagaa cctatgtaat actgcttttt ggtgctccgg gaaatttgta 57900 gccttaaaca cattattaaa caagaatgac aaggaatgca ttaaacatta aactaatagt 57960 ttagaaaagg gaacaaaata aatctcagga ataaataata atgtaaggag tcagtaaagc 58020 taaaggcaga aaataatgaa tttgaaaaca gtagaaatgg taaatccaaa actttggtgg 58080 gtgtcccccc ccctcccacc gacaaggaag tgagaagaga ggcaatgaaa tagataagct 58140 actaagtgct gccaggggga agggagagaa aacacagaaa aatgaggggg aaataccaga 58200 tactgaagaa tttaaattat tataaaggaa ccttttctgc aactctgaaa aatgttagaa 58260 tatccaaaga aattgataat tttctaggaa aacatgactt accaaaatta actctagaaa 58320 agaatcgata cacatcagta acaacagaag ttgagaaagt agttaacata ttgccaaacc 58380 tggaattcat gattgaattc tttgagatct actgtgagta catactctgc ctctgttcag 58440 ctgttccaga acttaagaag aggaacttcc aaattctttt ctcaaattca gaaacaatgc 58500 tattgaaata tattggaaac aggaacgaaa actacagttc agtttcactt ataatatcca 58560 ttctaaaatt gggaattaaa aatactagca tataggacta aatactggta catttatctt 58620 tgtttcccca aagattcact aaatgatggt aacggaatta aaaaggaggg gaaaagagtc 58680 caggaattga gtactgaggc attctcagga agagcaccaa gagagggagg agatacccag 58740 cggtcacagt ggaagtcgtc tttgagactt agagcgtttg aggaaggggt cgtctttaaa 58800 ataggttacc tacctagcac ttgggagacc agaccagcct gtggcaacca gtgacatcag 58860 ttcctttgag tctttccaga gatactctgt atattaagaa gcaaattttt ttttcctttt 58920 ttacacaaat gcattttctt gaaaattcct tctcatgaac tttgccagcc tctctgtctt 58980 ggtttgtctg agagggcctt tgatgcactc ttccatttag gtgctgtggt agttgaggca 59040 aagggacagt ttctgtagag tgatgaggga gtgacttagg tggcctggtc ttctgtccct 59100 tatctgtgta tctggagaag ggagcctgaa caccttcatt gtgatggttt agatcagttt 59160 tatgtctgta acccccgtgt tctcttttac atactcattt tactttcgta tcggttggta 59220 aaattgtctt tttttttttt tttttttact agagactaaa attctctctc tctttttttt 59280 ctacttgtca aaaggtaaaa ttttattttt ccagtggtaa tacttgacaa cccccaggtc 59340 ttggctattg ggacatgttt ctttacttct ctgataccgt agatttagtg tccttgatat 59400 ccccattgcc aggattccaa atctttgcct tcaaaggtat ttattctcct cttgtgtgcc 59460 agttccatag aggatgtata gatgtgaaaa tgtgtggagg atatagattg atcagtaagg 59520 agagaattct ttcaatttat acgtttccat gtaatttgat ttcttttttc caactaacat 59580 ctatttaaga ttgaaaaggg aatgtggaga aataagttgt attaagatag tagaaatagg 59640 ctgggcgcgg tggctcacgc ctgtaatccc agcactttgg gaggccgagg cgggcggatc 59700 acgaggtcag gagatcgaga ccatcctggc taacatggtg aaaccccgtc tctactaaaa 59760 atacaaaaat tagctggaca tgatggcggg cacctgtagt cccagctact aaggaggctg 59820 aggcaggaga atggtgtgaa cctgggaggc ggagctttca gtgagccaag atggcgccac 59880 tatactccag cctgggcgac agagcgagac tccatttcaa aaaaaaaaag tagaaataag 59940 gtgagtttct cttatttaaa tggcctttta caattacaaa actttaaaat ttttctggta 60000 tagtccttat agggactttc cttaaagttc acatttgcct ttcagtacat tattcctatc 60060 caaatttaac tatctttggt catcagattc tgagattatg aagtctctgg catttttatc 60120 tcacttgtat gcaaattgtc aacttcgtaa aaagctctgt gccagtaccc acctgttgct 60180 ctggactttt ctatatcagc agcttataat ttgttgattt ttttttttag ttaaatttct 60240 aaggtagcaa tgtggaatcc tgagatgaac gtagactttg gagtcaggca acccacaatg 60300 agaatcctgg ctccagcata tattagctga gtctttggat gagtttccta acctgtctca 60360 gtcgtctgtc tctgtatact ggagatgatc atactcagct gatagagttg tgaaaattaa 60420 gcttagataa gactgtgaat gtgaagcatc tggctctatg cgtcagacac agcaggcact 60480 agataactgt tagcttcctg cttcctccct gtcctgcttg tttaacttag cataaagctt 60540 atttacacat ttgctgcctg ccagtatcta cctaatacat aattccaata gagaggtgaa 60600 tacataaagt tatttgggct aatatcctgc cactacctct cttgggattc tttggctgct 60660 ctttttgcct acggaattaa atttttacta gtcttctatc tggtcccatt tacctgtcct 60720 ttttgttttc acattgcatc cctggtatga cctctgctgt aactcaggag ttctcgcatt 60780 ttaatatggt gtctgcagaa tattagttat ccactgctgc ctaacaaatt cctccagaac 60840 ttagcaactt aaagccaaat cttttgaatt cctgtaatag tccatctttt tcctgctttt 60900 tggctctaat tcactttgtt tcatttctgc tgtactgtta gtagagactt aaaaatgatt 60960 gccttcattt gacctgctcc cctgtcatca gaactgtgac aactctgtaa caagcattgt 61020 gctgcggagg atttaaaagc tatgtggtca ttagaatgct gaggctggct cagaccgcag 61080 ttgttggtag ttagtgcttt catgcaggca tttatcaaac aaatgcattt cagttgtgtg 61140 ccagccttgg tttcaggtat acagtagtga aaggagacag gcatgctccc tgtgggtgac 61200 acaaggataa caaacaggtg cacaaaaatg tgtcttcata gcttgaaaga catcccagga 61260 aagaagtgaa cagatgggct atgatgtaaa acaaggagac ctacttagat acgttttcag 61320 agaatctgcc tgagacttaa aggatgaaaa gggagaagac atttaaagga atttagtatg 61380 ttctgtggcc aggcgcggtg gctcacatct gtaatcccag cactttggag gccaaggcag 61440 acaaatcacc tgaggtcagg tgttcgagac cagcctggcc aacgtggcaa aaccccgtct 61500 ctgctaaaaa tacaaaaatt agccaggcat ggtgggacct acaatcctag ctgctctgga 61560 ggctgagaca gaagaatccc ttgaacctgg gaggcggagg ttgcagtgag ccgatattgt 61620 gccactgcat tccagcctgg gcatcagagt aagactccgt ctcaaaaata aataaataaa 61680 caaacagtat gttctagtaa ctaaaaggag ttggggtgac tagaacgtac ctttgagggg 61740 tatgcagaag ttggcaggtt gcagggatgc catggccatg gtcagggttt cattctcagt 61800 gctgtgtatc ctgagctata ttgatatggc atccaaagtg gagattgttg tctttttttt 61860 tttttttttt tttttttgag ttggagtctt gctctgtcac ccaggctgga gtgcagtggc 61920 atgatctcag ctcactgcaa cctccaggag gtgttgtaat ttagcagttg ggaagtaatt 61980 gggagatttg aattctagct accaattcaa ctataatgga ttggaaaaca agatttctgt 62040 gttagtttaa cagaggagag ctaaaaaaga actcaggcct gaatggttgt caggaattta 62100 cttgggtgac tatcttctca tctcagaaca taaaatgtag gaattaccac agttgcaaag 62160 ggagatgttt atgttggaga taaaaatgct cctcccttca aaaatgagac agtattttag 62220 ataggaaagg ttatttatct gattacatgt tttaaaattc tgagcgtaag gttatatgtc 62280 aaatcctgtc catgggctgg gcacagtggc ccacacctgt gatcctagca ctttggaagg 62340 ctgaggggga ggattgattg agcccaggag gtcaaggcta cagtgaacta tgatcacacc 62400 actgcacttc aacctgggca gcagagcgag accctgtctc aaaaaataaa aataaattaa 62460 caaaaaaatc tggtccatgt ccatctcctc ttagctgcta attcaatttt agattagaca 62520 cagtggacaa ggacaagtat ggtgagagtc ctgtgatttc tcaccagctt cctttccaca 62580 taggccgctg cttctcttct tccaaggttt ttccccgctt ttgcctcctg gaggttgtat 62640 cctgggtgtt aggagactgg gttccggaca cattccccac agaaggatag caggacctta 62700 gaagatcttt ttctttcttt tcctggtttc ctcttgtttg caagagggtt gaataggatg 62760 gtctctaaaa tcctgttgtt tttctgggtt atattaaccc aggccataat gataagaacc 62820 tgctctgaat tcacaacatg tatttataca acagcaattt aatatttctt attctgtgga 62880 atggctagga agctctgctg gtcttggttg gatggttttt tgtttgtttt tttttgagac 62940 agggtctcgc tttgtcgccc aggctggagt gcagtgacgc catcagctct ctgcaacctc 63000 cacttcccag gctcaagtga ttctcttgct cagcctcccg agtagctggg actacagaca 63060 catgctactg cacccagcta atttctgtat ttttagtaga gatggggttt caccatgttg 63120 cccaggctgg tctcgacctc ctgagctcaa gtgatccacc caccttggtt tcccaaagtg 63180 ctgggattac aggtgtgagc caccgtgctc ggctggtttt tcttaaggtc tcacctgggt 63240 tcacttgtgt ggctgaattc agctggtggt ttggcagggg ctggatgcag ttacaacaga 63300 ggatctgtct ctttaaataa ctacccttca tccccaaaga ggccagacca gctccttcac 63360 agtgctccaa gagagcaagc catgacgccc aagcatttta tccagcctct gcttgcttca 63420 gtttgctaag gtcccactgg ccaaagcaga ttacatggcc aggtctaatg tcaatatgaa 63480 gggggcactc cacaaaagcg tgaacatgta aggcatgact catgagggtc actaatgtaa 63540 tagtcaccac aacctccatg ctaagttacc tatattctcc agtgaggatt tctcaaggtg 63600 gttttgttca tagtcttcta atagaattat ttggaattat cagtttaata tgcttatgat 63660 gtatttcact ggaaccatac aggttttgat tcgcagagtt gggagccctg ggtagatact 63720 gaatcagact aagtttaatc acagaaatta ttcctgcgta agtctgatct tatgttttca 63780 agatagcatt gtaaaattca gagtatgtta ccatccccct ttgagacctc tgctgttttt 63840 aataaatgga agcatttggg aatactattt ggtaatagtt tattaaaact acttcagaga 63900 tattctggac tttcatatta gtcttagata tggattaata aacattagca atgaatctgt 63960 tatctaagag aaaaatttaa atttatatta caacagtgga atataatgtt taataacttg 64020 tgttgggggg attatgtgtt ttgtttgttt ttttttttag ctcttcagag aagtaagaat 64080 aatgaagatt ttaaatcatc ccaatatagg tactttctgc ttttttaaat attttggggt 64140 ctaaatacgt acttgaaatt atgtcataaa gctaaacacg tattctagaa atggtagagt 64200 acacttctag taaaatatat atacaagttg ttgatcattt gtattagctt tttgaaattg 64260 ctgaagacag gttaaaagct taggtattaa acgttgaatt taaagcttta atctggtaga 64320 aacatctgta ctctgattat aattttctaa tttttaagta tattagaaaa tataattgta 64380 ttgcatgagt agatagaagg gaattatagg aagtcagaat taatattttc aaaggggctg 64440 ggcacagtgg ctcatgcctg taattccagc actttgggaa accaaggcag gaggattgct 64500 ggaggtcagg agctcaagac cagcctaagc aacagagcga gaccccgtct ctccaaaaaa 64560 aaaaaaaaaa aaagtaataa taataataaa cttaaaaatt tgtaaaaaga atatttcaga 64620 ggtccaatac tttttgctgt gtgccctaag aaaatactta tttgaaagat ggaatacttg 64680 ctatctaatg gaattgtgat aggaattatt ttataaatca aagatttgtt ttctgtgtcc 64740 tctgtgtgca caactctgtg ctgggtgcta gtaggtgtgt ttaaagatga ggaaggagcg 64800 acatggtcct acccttagag taactgtaga aacaagaaga gaataagcaa atgacttaat 64860 actgacccaa gaaaaacttt tgatcccata tgcattgtaa tagggctttt aaaaaattac 64920 ataactgctt ttttgtatag tgatagatca tacaatctaa aaataatatt tcaagaatga 64980 aatcactctt aagacagccc ataatcagct taactgtcaa catcagtttt aggaaatgaa 65040 aggattgatg tttagtatca aggatagcct atcaagaatg catcaggcac aagaatagaa 65100 gagtaacaca gaaccaccac aggaagaaag aagcttttac agagagctgc ttttttaaca 65160 aaaggcgtat gccctatttc atcagtctaa accaccatta ctttaaaggt gttcttgttt 65220 ccttgtttca tatactacta agagaaatgc tagcaagcct tcatcctgat atcagggata 65280 ttaattaaaa tgtgaaaaaa aaatttagaa tcaataagta tggttgaaga aaaaaccacg 65340 gaacaacttc atagttggat taaaaaaaaa tcacaaggaa tataatagtg ggaagaaaat 65400 cttgtttcct tgattttcat ttcaatcctt tggggctagc tagccaactc tggatttcaa 65460 attccaactt ttcacacccg tcctcccgcc ccccacaaaa aaactttatc actgttgcct 65520 agaacaagct aacgtaaaca tgtttatttt gtcttttaat tacttaaatt gtgacctgat 65580 tagagttttg tacttaaaac ttgacatatc tttgataata aattgaactt ttaaaaaatt 65640 cctattgcat taacatagtt ttcccagaag acccaaagtt tcgttggaag attagaagag 65700 ttttattttc atgcagctta ccaacacatg tgccttaact ttctgaagtg gcttttcttc 65760 acagtctgaa cgtatcagag tctagggaat ggtatgatag ctttattcat cagttcatca 65820 aacatttact gactgctatg ttaggcattt tgtttggcct gatactctat taaagcctca 65880 gaaaattgct tgaaatataa aacactagca taccccccag ttttgggtaa acttaaagta 65940 aatattaaca taataaagta gatatgcaca atggtgattt gatagcttca gggatttact 66000 ccagtttcat ttttaaagtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgta 66060 ttcttttttt tttttttttt tttttttttt gagacagggt ctagctctgt tgtccaggct 66120 ggagtgcagt ggcatgatct caggtcactg caacctccac cacactggct caggccattc 66180 tcccacctca gcctcccgag tagctgggac tacaggtgca tgccgccata cccagctaat 66240 ttttggcatt tttttgtaga gagggaattt tgccatgttg ccgagggtgg tgtccaactc 66300 ctcagcagaa acgatccacc cgcctcagcc tcccaaagtg ctgggattac aggtgtgagc 66360 caccatactc agccaaaaat gtatatattt ctaataaggt tttatgaatt agcagtgata 66420 gaaatatttc catctgtaac aaaagactgc tgtaggaaaa tcaccctgac ctactgaaaa 66480 tgattcttat aaaaaagatt ccccccctca attaattgca gtataatccc tctacttctt 66540 tccatctttg gcatcagaaa agtaacaaag gaaccttgtt ctttgaaagt tgtcataagt 66600 ttcccaagca ataaaggtct caaatagaat tacatcctta aagccataat cataagcagc 66660 tagatttgca tttgttggag cagagtagaa ctgagcagtt gctgcaggct gaccactttt 66720 cctggggtgc tgggagggca gctagccaac acagacatgc tgaaggacag tgagggtgac 66780 agaggaagtg agctcaggta caccttgctg gactgctgag cacatatgga agtcacactg 66840 aacattcaga aattattttt atggaattcc atgctttcat agactctttt ctgttgttgt 66900 ggtatttgat aaaattccct aaaagcattt tttagagggc cagctattaa aatctttaac 66960 agggaaaagg ttgcttttca tagttagagt ttatatgtgc atggtttgtg catacagaca 67020 tttgtctctt tttcttccgt gtcctctcct ctcccgcagt gaagttattc gaagtcattg 67080 aaactgaaaa aacactctac ctaatcatgg aatatgcaag tggaggtaag aacattttta 67140 tatatattgg gttttttttc tttctccctt ttaaaaaaat acacaaccat actgcccata 67200 tgggtcatca ttaaggtctc atttaacgtc cagagccata atacgctagg atgagagtcg 67260 gaaaagctga ctcttagcac ttctaggggt tgccatgaag tgtttcacta ttagcattgt 67320 taattggtaa tatctaaata cctggatatt ttttgtggta aaacatgcat cactgaaaat 67380 tatcgttaaa atcattttta ggcgcacagt tcagtgacat tagcatgttc acgttgccct 67440 caccaccacc catgtccaga atgttttcat tttctaacac gaaactctat acccattaaa 67500 cactaactct ccatttctcc ttctcccagt ccttggcaac cattctcctt tctgtctcta 67560 tgaatttgac tactcttgga acctcataca agtggaattt tacaggattt gtcttttttt 67620 tttttttttt tgagacggag tctcgctctg tcgcccaggc tggagtgcag tggcgcgatc 67680 tcggctcact gcaagctccg cctcccgggt tcacgccatt ctcctgcctc agcctcccga 67740 gtagctggga ctacaggcgc ccgctactac gcccggctaa ttttttgtat ttttagtaga 67800 gacggggttt caccgtgtta gccaggatgg tctcgatctc ctgacctcgt gatccgcccg 67860 cctcggcctc ccaaagtgct gggattacag gcgtgagcca ccgcgcccgg ccaggatttg 67920 tctttttgtg tctggctgat tgatgcagca tagtgtcctc aaggttcatc catgctgtag 67980 catgtgtcag aatttcattc ctttttaagg ccgaataata ctccattgtg tgtgtgggac 68040 acacacctca cattttgttt atcttgagta tgtggctatt taaacatatg aatgcttagt 68100 ctgtttgaaa caaatgtgtg ctttggttta gatgcttttc tttaccagat tttaatgtcg 68160 ctggtgtctg tcttccccaa ggccagaaat gatggttaca gtacacatca ctagagtttc 68220 cttaaaataa agattaatga ctagtaacta tttgcctatg gttttgtaaa aatgtagaca 68280 ttttctgaaa tgcgtgttta tagctgctgt cttttataat gatttgtatt ttatggttga 68340 gattgggctg ggtttgtagt ttgcgaccac acgtgagttt cattgtctgt gaagggcaga 68400 agctttcttg ttcatctttt tgtgtcccct gcctctagca cactgcctgg cacacagcag 68460 atactcaaca gataagaatt agactgcatt taggaattat aaactactgg gtacacattc 68520 tgttaaactc tatcgtaatt ttatcattag cactttgatc catgttacaa aacctgaaga 68580 tagaaagttg gattatagtc tcatttgagt gagtttacca ttgaaaataa aaagattgta 68640 aacctgttgt ggaaaacaat gagttgtagt aagcatacct ttgacaccac ttttttatac 68700 tcctaattca ttattagttg tgtattttat actttatata tgtctagttt gggaatttca 68760 ttgggatttt caaaacttca ggggtagtag aaagagggga aggttaattt caggaccaaa 68820 aagctttatg gagttctaat actttctgtg ggcaaacaac acagagtaat gttcatagcc 68880 ctcacgttgt acagcctcta cagtgtacaa ggtgctttct cttaccagat ctcctttgac 68940 cttcacagcg actccatgct gtggccaggc agtgagcgat gggcttttta cccatgagga 69000 aatggaggct gggaagtctc actgtgggcg ctctgggcct ggaccgccag tgctctgaca 69060 gcagatagcc tttctagttt gttggtcagt cacggctttc tgttcccatc tgttttagct 69120 acccaggtca cagagattac tcatataggg gcaagacaaa aacatctaag agtcatccag 69180 gtttagtaga aagaggatgg gctctggaag agacagacat ggagtgaatc cagccagtgg 69240 ccctcattgg ccatgtgacc tggcaagtaa catgtgctga gctgagcttc acggtgagca 69300 taggaacccc ctctgagggc tcagtgcact tggcaacatt gtaagagcct ttaatcattt 69360 aatcgaaggt ggtggttctg tattaccttg ggtttttttt tttttctttt tggagacagg 69420 gtctcactct gttgcccagg ctcaagtgca gtggcgccat ctcagctcac tgcaacctct 69480 gcttcctggg ttcaagcaat tctcctgcct cagcttcctg agtagctggg actgcaggcg 69540 cacaccacac ctggctaatt tttctaattt ttatagagac aaggttttgc cacattggcc 69600 aggctggtct tgaactcctg acctcaagtg atccacctgc cttggcctcc caaattgctg 69660 ggattatagg tgtgagccac agcacctggc ctctttaaca gtgttttgtt gagtttatta 69720 aaacaatttc cgggacatat gttttattgt tgatgtgttt gccattgtga aaagttttat 69780 taaattggcc acccattcca ccattgcatc tcccccaccc gccagcctgc tgccttttga 69840 tttggtaaac tcatagaatt ttagaattga aaataatctt agaaatttta gggcagcggt 69900 ctaattttta cagatgaaac tgaagctcag aaagtttgtt ctatgccaag gggtccatag 69960 ctagttagtt tcaggacctg aactagaact aagggctttc tgaactggcc tgtcagtgtc 70020 cttccatcca gccacctgtt cctgcccagg caggagagcc actctttgct tcttgtttct 70080 tttatctcta ataaatagcc ttagtatttt tcagttcagc tgcttaacct gaatgttaat 70140 acatttttaa taaggaaaaa agatctggat tgaattcctg gtttaaaagt tgaactcctg 70200 aattataatt tagtaattat gagtgtgaca tatggttcca caaatctctt aagaggtttg 70260 tattgaattc aaatttagaa aaaaaaatct gtcaattata ttgacagact tggattttat 70320 ctgtgttact ctacaacagc tggtaggctt aatcgtttaa tttttttaag tgaaaactct 70380 cctatatgat attcactcat gtttagttgt ttttgcttat taaccacttg ttttgacatt 70440 gtgtgctttt ctgcaaatag gtcattcgca tagaaaatgc tgacacttta ccgagctgac 70500 atttaacttc ataattcatc atagttaagt gaattgtgtc gtgtaaactt gacagtatgt 70560 aatgcctttt aaaagatcat tatgcaggct gggcacggtg gctcacgcgt gtaatcccag 70620 cactttggga ggccaagacg ggcagatcac ttgaggccag gagttcgaga ccagcctggc 70680 caacgtggtg aaacccccat ctctactaaa aatacaaaaa ttagccgggt gtggtgacgc 70740 acgcctgtaa tcccagctac tcaggaggct gaggcacgag aattgcttga acgttggagg 70800 cagaggttgc agtgagccaa gatcgggcca ctgcactaca gcctgggcaa cagcacaact 70860 ctgtctcaaa aaaaaaaaaa aaaaagcatt atgcaatcaa gtaataacat gaaaatattt 70920 gtgcccattc attatgtaaa atttattctt tcagagttag ggttaataag agtttcaaag 70980 tcagataatt gtgtaattca tgatgacttt caagtatcaa aatattttag tttaatattt 71040 tcactaagct gatggaggta ttccttattt gtatgaagta aagatgtttc ctgaaaacac 71100 ttatatctaa ttttctaaat tagtattctt ttctattgat ttcagaggtg gtgattcttt 71160 attctacatt gataagcagt tgacagtgct aataatatat tccttgaagt gtcacctttc 71220 ttccctaaat aattaatgtt gtgtaaactg ccacctaggc gtgcatcagc tggttctgtt 71280 gttttcacct ccattgtatc ctgagctcct acttctcacc accactgtgc ttcccactct 71340 agtccccgtc actttcatga ttggtttgat tattgtactg atctccctct tggtcttcct 71400 gtcccttccc gtcattggaa gcctcttccc cgctagcagc cagtgatcct tttaagaggt 71460 cagtcttttt tcatctctgc ttctcatctc acttgtagta gagccagagt cctcaccacg 71520 gcctacagga ctcttcctag cctcatgaac taccctcttc ccctcattca cacttctcca 71580 gccctgtggc cttcttgctc tttctcttaa ttactgtgga atcttacccc agataactac 71640 attgtccaca ccctcaagta gtcagcaaaa cacaagggtg tgcacacaca ggctcacttg 71700 tctgtctctc ctactttttc tccacagcac ttactgtcct ctgatacact atatgtttat 71760 tcatttattg ccccctcccc caactagaat gtaaactcta tgaggaaaag gatcttgtgt 71820 tcactgctgc atctccccag aacctaccta gaacagtgcc ttgcagttag tagacattca 71880 ggaaatattt gttgaatgaa tgaatatact caggaaatgc tttgttgtca taatcctgca 71940 gtgaggatgt cctcttctaa cacaaataac ttcatccatt ttaattttct gttttaattg 72000 cttagttttt attaaagcct attgaaaacg ctctttaaaa taagagttat ataatttaag 72060 tatagggaat ttaattttaa ggcttttctt cagcttaaag attttgttgg tgaatttaaa 72120 tgcctgtagt taaagccagc ttagttcaaa ttccacatat ttctggctaa ctttatatct 72180 atatttaaaa attagagcat tgctaaaagt gaaacatcaa tttatgggaa aattaatact 72240 cagaagtagg atttctactt acttttattt ctctcaccta taggtgaagt atttgactat 72300 ttggttgcac atggcaggat gaaggaaaaa gaagcaagat ctaaatttag acaggtatga 72360 attaatgtgt ctttactatg tcaatttgat aatttatctc acttaaaccc tgaagcaaac 72420 aagtgtttgc ctccataaat gcttataagg cctgttggat ggcaggggtt ggccattcaa 72480 ttcaacaaaa attgatgaag aacttttcat acctaaggca ctgtgctgga ggcagccatg 72540 gttctattcc tagctttgta atgggagcat tagtcttata cttaaccttc cctttttaca 72600 actgagcctt aaaaatctag agcctttcaa aaacacctgt gattacatta attaaagcca 72660 tttcagagtt tttagcaagc agaatgtcag aaccccaaaa ttcattatta gctttgtctg 72720 acataaacca aggccaagtg taactgaaac tgttaattag taactttact tcttgctttg 72780 tttttactct gctttttaaa gagactcggg ttttaataag caggttttaa gcaaacaggt 72840 tacttgactc tcctgtcttt attaataata attactgtta tctattgacc atgccacaca 72900 ctgagataag tactttacca acattaaggt aaatcttaca gctgccttgt gagttaagga 72960 ctgttatatc catttgctaa aaaataagac aactgagaca tgggaagatt aaataacttg 73020 cccagaattg cctttctttt tttctttttc ccccttattg tggagaacgg ggtctcgctg 73080 tattgcccag gcaggtccca gaattgcctt tcaagtagga gacctgccat agactcagag 73140 tcccaaaccc tctgacccca aaactcagat ttgcaatcat tatattgtgc tattatttag 73200 actgggaatc agcaaacttc tgtaaagggc cagatagtga acattttagg ctttgtgggc 73260 cacactgtct ctgtcgcaac tatttaactc tcttgttgta gcttagaagc agtcgtaggc 73320 tgggtgcgat ggctcatgcc tgtaatccca gcactttggg aggctgaggt gggcggatca 73380 cctgagggtc aggagttcga gaccagcctg gtcaacatgg tgaaaaccct gtctctacaa 73440 aaacacaaaa attagccggg catgatggca ggtgcctgta atcccagcta ctggggaggc 73500 tgaggcagga gaatcgcttg aacctgggag gcggaaattg tagtgagcca agaccgtacc 73560 attgcacttc agcctgggtg acagagactc catctcaaaa aaaaaaaaaa aaaaaaagaa 73620 gcagcagctg tagacaatac caaatgaatg aacgtgactg tgttccaaca aaactttatt 73680 tacaaaaaca gggatgggcc ggatgtagcc agaggccata atttgccaac ccctgattta 73740 gacgaaggaa aggagcagtg cttcactgct tttaaattaa ttctgtattc tcacaaggcc 73800 tacattgaaa tggaattata gcctcatttt ttcttagaac ctttatattt tgttttattc 73860 atatacaggg ttgtcaagct ggacagacta ttaaagttca agtctccttt gatttgctta 73920 gtctgatgtt tacatttgta agtgatagga cttattaagt ttcttataaa cgttgcttat 73980 attttgctgt tgcttaaata ctaatggtac tttgaattca aatctagtaa aaccaaagta 74040 aaaatcagct ttggctatca tttaacttct ctgatcctgt ttttaaagct ataaaaaaaa 74100 aagaaattat tcttgatgaa ttccatagtt ctttgcaatt ctaatataat ttgattgtat 74160 ggtctattaa aggaaattca gatttttatt agaaaaaaag tgtgtggctc tttccataac 74220 tgtactctta atttttataa attggccacc taaaagggaa catttttttg cttcatacaa 74280 tttacattcc ttcctactca gatgaatctg acttgcaaac atgtgtgaag atggcttatt 74340 gactatgagg aaggggctgg tgtcacccag caagagctta ctaacctaaa tcttgaggaa 74400 attactctaa tttttattgt aaattccctg cagaaattct gaagccttta tttgaggagc 74460 ctgttagttg gactaggaaa gatggtttgt atgtatgtgt tttttcaatt agcaaattga 74520 tttagagtgc tttagaattg accctttctt ccatggtatt tgcctaaaac agctccatta 74580 gacttggaag gatacgcatc atattggtgt ttccactttt ctgtttcaaa tgctgtggtt 74640 tctttgtttt ttcgtttttg agatggagtc tccctctgtc accaaggctg gagtgcagtg 74700 gcccgatctc agctcactgc aacctccgcc tcccaggttc aagcaattct cctgcctcag 74760 cctcccaagt agctgggatt acaagtgtgc accaccacgc ccagctaatt ttttattttt 74820 ttttttgtaa ttttagtaga gacaaggttt caccagtgtt ggccaggctg gtctcagact 74880 cctgatctca agtgatctgc cctcctcagc ctcccaaaat gctgggatta cagatgtgag 74940 ccatcgtaat gtggcctcaa tttctgtgct ttctaggagc ttataagtca taattacttt 75000 gactaagtaa aacaagtttt tctatttatg acaaaaagga aggtatgcca cacacaaagt 75060 acactgtgtg tgatcccttt tccatattca tcaccagatg ggttttcccc ttctgcttcc 75120 ttcaccatgc ccatcctagt tactgcttat gacattttaa ttttttggtt gagctcattc 75180 ttttccaaga aaaagaatag atttccagct ccatcttttt tttttttttt tttttttttt 75240 ttttttgaga caaatctcgc tttgtcgccc aggctggagt gcagtggcgc gatctcggct 75300 cactgcaagc tccgccccct gggttcacgc cattcttctg cctcagcctc ccaagtagct 75360 gggaatacag atgcctgcca ctacgcccgg ctaatttttt gtatatttag tagagacagg 75420 atttcactgt gttagtcagg atggtctcga tctcctgacc tcgtgatcca cccgcctcgg 75480 cctcccaaag tgctgggatt acaggcgtga gccacgcgcc cggcacagct ccatcttttt 75540 tacttacctc tttattggtg ggctggtaat attgatgatt tgacgctctt ttatgtatga 75600 ttgaaagtga attaggttaa atcatagttt aacctaaata acatctgtta gaaatcacca 75660 tttctacctg gtaatagaat gcaaaaaaag cagtaagttc aagttgagtc ttttcagctt 75720 attgtggcat caggttagct ttgagctgtt ttggagctaa agagagagag aaatcctttg 75780 atctctttga gaaagggcag gaaatcaaat ctgcttcaaa agcagaattt gaaaataacc 75840 tgtaagagaa atctcaatat gaggaaaaac taatataaat tctgaatgag aaaaggaaaa 75900 agattagata aaatttacct gtgtcctagg tgacaaagct atgactttgt tttgggaaaa 75960 caaagctatt ttacttttca agttttcatg ctgactgaat agaacaatag tgataaatcc 76020 aaagtaacag atgctctaaa acccgttttt tggcattgga atttttgtac cttttgttaa 76080 ttataagtgg gtctgatatt ctgaatgtta attttaaata atgaagctgc ttagtaaagt 76140 gctgattttt gttatcatct catcctaacc ccagttcacc ttgtccttgc tgcttattct 76200 gaaccaaaat attatacagc cctctgagat tttatgtcca cgggtgggtc catggggctt 76260 agagctaaga gttgatgatg atttggagga accaacagta attttattat aacctcagtt 76320 ttgtgtatat gtaatcatat gtgtctaaat atacatgcag aataccaaag aaaagactag 76380 atgcaagtac agcaaaatag caagacatgt tacctctgct tagtggtaat gcaggaatat 76440 atttctatag acttttctgt atttcctgaa atttctacaa tgaccatata aagctttaat 76500 attagaagac tttttttaag tgaccgtaag tgtgaaattt cagtaaaaca attgtttaat 76560 cataggcatg actgcaagtt gactaaataa aagcatacta tttactgaaa gtgggaggaa 76620 tccttaatag tgcacgtagt agttttcata gttcacgtac gtcgcaacat tcattcctcc 76680 attttaataa aggaagagga aactgaccct cagagaagtt aactaacttt tgctttccat 76740 gatccacatc agttagaagg aagactgttg cagggagcag gctgagaagg gcacttacag 76800 aagagagcca acctggatta acacctgggt atgggcaact ctctaaccgc tggtttggtt 76860 tggagttgac tctatgggta tggaaaggtt gcaccaggcc gggcgcagtg gctcacacct 76920 gtaatcctag cactttggga ggctgagact ggcggatcac ctgaggtcag gagttcgaga 76980 ccagcctggc caacgtggtg aaaccccatc tctactaaaa atacgaaatt ggccgggcat 77040 ggtggtgcac acttgtaatc ccagctactc gggagactga ggcaggagaa ctgcttgaac 77100 ctgggacggg gaggttgcag tgagccaaga tcatgccatt gcactctagc ctgggtgaca 77160 gaacgagact ctgttaaaaa aaaaaaaaaa aaaaagttgc accagagcca tcggagccta 77220 ggggcagcca gggtcctcca gatctaggca atttgttggg tcctggcact acactgtaag 77280 tggaatgtca acggataggg tccaaaaaga gacttcagtt aaaacagtag aaaacggaag 77340 aacgtcctca cctatttgtt tttgccctca cccatgaatt tctccagact ggaaagttca 77400 aggcatagga aaattgtttt tagaaatcca cacggacatt cgagcgtata ctgagtattc 77460 attatgtgtg aacattaaca aagttatttt catcttaatt acgaatctgc accagtgatg 77520 actgactctg cgtctctcct attcagattg tgtctgcagt tcaatactgc catcagaaac 77580 ggatcgtaca tcgagacctc aaggtgagta gaagtgcctc actcagtgta tgctctgtct 77640 gtttgtgtgc agttctctca gtggtcatta cacaaatgag gtatagatta gccttattag 77700 cattttttaa aatcccacaa taattgaatc ctcttaatca agttatggga aagagcatta 77760 aaaataaaga aaaataaagg ttattgaaag tattttacat ttcagttacg gtgactggtg 77820 gttgatctct ggagaacagt catcacacag ccaaacaaat gtgggcctcc agaaatgtat 77880 ggtcacgccc acagcatcct cagtgccacc cggcattcat ttctgctgct ttcatcagta 77940 ttctctcccg ttaacagcag tgcctgtttg agacctcttt caaggtcctc agcctctgcc 78000 ctcactctgg cataggatga ggtttcgtat cgcacagaga aaatagaaac accatgcaga 78060 aactctcatt ttctaccagg ccctcaaacc tagcagcacc tacccccatc catcacttct 78120 ttcctcctgt tacaggaaag tggggtgctt ctgctctgag ctctgggtcc tgattccctc 78180 tgggggctgt gctctgtctg atgcctcttt tctctccgtt tgtttattca ctctccttct 78240 ctactggctt ttcaccagca gcatttacac atgctcaagt ctatatacct tttttttttt 78300 ttaactaaaa agccatgcct gttcctcttt cagatacacg ctttttctct cttccctttc 78360 atagccatat ttctctaaac atttcctaga ttctgtgtct taacttggca ctcactttca 78420 ctctcccctt acgtaattgc cattgacttg cttgttctct gcacccgccc ctagagcatt 78480 cacattcact ctctcaactg cagtggccaa ctgtatgctg atggccccaa atccattcct 78540 acggccagag cactcttctg ggatccagga cccagctgag caaagggtgt cttcatataa 78600 gcatcccttg ggtgcagcag gttcaaatcc tataaacatg aatgttcact tctcattcag 78660 gtgtctgatg tccctcaagt accttactct cctgtgctac ccactcagtc accaggtcct 78720 tttgctgctg tgttccaagt atctcttaag ctcactcttt tatgtccctg ttacactatc 78780 caaggccaga ccaccattat ctcataggtg attccgacac tactttctta tatgttcttc 78840 tctgtttcta gtgcctcttc ctcccatgta tgttttctac actgcaccca aaatggtctt 78900 ttaaggaaac acaaataaga tggtcatttc tctgcttgaa caactcttca ttggcttccc 78960 attgctctta cgatgaaaac ccaaacttct caagtacaat cctttgtgat taagccctga 79020 ccatctcccc agccccctct acacatctct atctcacata ctgtctttca atttcttgtg 79080 tgaaacatac tgtgtctcac atgggttctc agactcttta ttctgcctga aatacccctc 79140 atcaacacat ccattatgat cagatgcacc ttctggtgct ttgatttcac ttcatctggg 79200 aagccattca cacctttcag tgccaaccca caactccctg tgcctcttca gttatggcat 79260 tttgtctcag gacagctgct tatcattctc cttcttccct tctaaactgt gagctactga 79320 gggtagggct ttatctgtct tgttcatcat tatccatcca gacactaata tagcatctgc 79380 tgtggagtag ccacatagga aatgtttctt aaagtgactg tgatttgtct cttttttttt 79440 tttttttttt ttttttttga gacggagtct cactttgttg ccctggctag agtgcagtgg 79500 tgcgatctct gctcactgca acctccacct cgcaggttca agcgattctc ctgcctcagc 79560 ctctcaagta gctgggatta caggcgccca ccaccatgcc cagctaattt tttgtatttt 79620 tagtagagat ggggcttcac catgttggcc agactggtct cgaactcctg acctcaggtg 79680 atccacccgc ctcagcctcc caaagtgctg tgattacagg cgtgagccac cgcacccagc 79740 catgatttga ctcttgaatg aggtttagga ttctcccctc ctcattaaca gccgttttat 79800 agctatttag ccgtttttat gaaatgcccc tttttaaaaa aagttattta tgttttgaga 79860 cagggtttta ctctgtcacc caggctggag tgcagtggca cagtcatagc tcactgcagc 79920 ctcaaactcc caggcttaag aggtcctcct acctcagcct tccaagtagc tgggattgca 79980 gatgtgtgcc accataccca gctaattttt aaaaatcttt tgtagagaaa gggtctcact 80040 atgttgccca agctggtctt gaactcctgg gctcaagtga gccactgtac ctggccctgc 80100 cccatattat tcttgttttt tggttttgag atggagtatt gctctgtcgc ccaggctgga 80160 gtgcagtggc acgatcttgg ctcactgcaa cctctgcctc ccgagttcaa gcaattctcc 80220 tgcctcagcc tcctgagtag ctgggattac aggtgtgtac caccacaccc agctgatttt 80280 tgtattttta gtaaagacag ggtatcacca tgttggccag gctggtcttg aactcctggc 80340 ctcagatgat tcgtccacct cggcctccca aagtgctggg atcataggtg tgagccaccg 80400 tgcccagccc ccatattatt attatcaatt gaaaaactca aggcttttct tgatcttggg 80460 acattgatgt ttaaattagt gctagaatta atctgtactt catgtttgtt attgttaata 80520 ttataaaata aggagttctg acttgtctcc tgtttttttc tctagaaaga atgttttcac 80580 attaattagg aggtaacttc atatcattac agaaagcttt tctaaaatgc ctaatcctgt 80640 cataattcta attctatttt ggctaaattt catttttgtc ttgatgtttt ccctctaggc 80700 tgaaaatcta ttgttagatg ccgatatgaa cattaaaata gcagatttcg gttttagcaa 80760 tgaatttact gttggcggta aactcgacac gttttgtggc agtcctccat acgcagcacc 80820 tgagctcttc cagggcaaga aatatgacgg gccagaagtg gatgtgtgga gtctgggggt 80880 cattttatac acactagtca gtggctcact tccctttgat gggcaaaacc taaaggtata 80940 agaagctgca cccatgtact tcactaaact aaaagaagtt tcctaatatt acatggctta 81000 atatttttaa cattatatca gtgcgggggg tttcaggggg ttttttgttg tgttttgtta 81060 actaaaccta aaggttgact tactcgtttt ctttcctctg tacctctcca aaggaactga 81120 gagagagagt attaagaggg aaatacagaa ttcccttcta catgtctaca gactgtgaaa 81180 accttctcaa acgtttcctg gtgctaaatc caattaaacg cggcactcta gaggtaatca 81240 tgtaggtgga aacaagcagt aactttggag agtctttaga gtgaccttag atctttgctt 81300 gatttgtatg ccatactgga tatatcctgc ggctttttaa gcaagaattg aaacattaaa 81360 aaatattttt tgagtttatg ctttgaacga tagtcaatga aatgttgaaa ataaattttt 81420 gtaaatatta cggttatcag aatatttcat tttactctgc taatgaacag tttacctttt 81480 ttagcaaatc atgaaggaca ggtggatcaa tgcagggcat gaagaagatg aactcaaacc 81540 atttgttgaa ccagagctag acatctcaga ccaaaaaaga ataggtaatc actccatgcc 81600 tgcatgttca tgtgtttttg tctaagtaac atatttctgt tttgactcat gtctgtgcct 81660 aaaatgtgaa taatggaaag ttaagcacaa gtcatatgaa cagtttatct gttctgtcat 81720 atttaggaac gtaactacct gcagtttcct ataatggcac ccagtaactc tgaaacaagt 81780 gcccatttat gttacaaaat atatgtaata tgtcatcttt taggtcaaat gaaaattatt 81840 ttaccctaca aaagaatgat ttctggccgg gcgtggtggc tcacgcctgt aatcccagca 81900 ctttgggagg ccaaggcggg cagatcacct gaggtcggga ggtcgaggcc agcctgacca 81960 acatggagaa accccatctc tactaaaact acaaaattag ccaggcacgg tggctcatgc 82020 ctgtaatccc agctactcgg gaggctgagg caggagaatt gcttgaatct gggaggtgga 82080 ggttgcggtg agccaagatc gcgccattgc actccagcct gggcaacaag agtgaaactc 82140 cgtctcaaaa aaaaaaaaaa aaaaaaggat gatttcttac ccaaacaaaa cacataaact 82200 gtattatgcc cttcttttag atattatggt gggaatggga tattcacaag aagaaattca 82260 agaatctctt agtaagatga aatacgatga aatcacagct acatatttgt tattggggag 82320 aaaatcttca gaggtaagag taatcagaaa gagctgaaat accctgtatg taattattag 82380 tttatataaa ctgttttctt agtttttatc ttttgaatat ttgtaacatt tgatacatca 82440 ttttttagat ttacttgcaa atagcaaatc taaatcctat gactattata agctatttta 82500 acttaaccct taatgatgga tattggccag gcgtggtggc tcatgcctgt aatcccagca 82560 ctttgggagg ccaaggcgga cagatcactt gagaccagga gttcgagacc agactggcca 82620 acatagggaa actctgtctc tgctaaaaca aacaaacaaa caaaaaatta gctgggcgtg 82680 ttggcgcttg cctgtaatcc ctgctccttg ggaggctgag gcaagagaac tgcttgaccc 82740 tgagaggcag aggttgcggt gagccaggat cacgccactg cactccagcc tgggtgacag 82800 agcgagactc tgtctcaaaa aaaaaaaaaa aaaaaaaaag atggatgttg tcataaacct 82860 ggtctttcat aaattaacct gtagtcatta atagcttaga aatgacgtaa agcatcagat 82920 gaagtaaata caacatttaa agttatttga gaaagcctgg aaagaaaatc ataaatacct 82980 tacctttagt gtctttccag ccgtcagaaa actacttttg cttgagttta gggttgtcat 83040 ttttgtacgt tgtgattcct cctctctctg aatgaacggt ttatgagagg tctgtgttaa 83100 tgaaaagttt aacttcctaa taagccattt gggttcgtgt tgtggtttgc tctgtttttc 83160 tcattttctc ttagctggat gctagtgatt ccagttctag cagcaatctt tcacttgcta 83220 aggttaggcc gagcagtgat ctcaacaaca gtactggcca gtctcctcac cacaaagtgc 83280 agagaagtgt tttttcaagc caaaagcaaa gacgctacag tgaccatggt aagttttgga 83340 gtatcccagt gccttctctt agagtccagg caagaggtct cctagcactg ggaagcattc 83400 tcttgctcag agcctggctt gatgtccttt cttggcattg ctttctttct ttcttctttt 83460 tttttttttt tttttttttt ttttgagaca gagtctcact ctgttgccca ggctggagtg 83520 gagtggcaca attttggctc actgcaacct ccacctccca ggttcaagcg attctcctgc 83580 ctcagcctcc tgaatagctg ggactacagg tgtgtaccac catgcctggc taagttttgt 83640 atttttagta gagacggggt ttcaccacat ttggccaggc tggtcttcag ctcctggcct 83700 taagtgatcc acctgccttg gcctcccaga gcgctggaat tacaggcatg agccaccgtg 83760 cctggccagc attttttctt agtgttcctg caggtgctgc catgattaca tattacttca 83820 aggctcttat cttctttata catgcagtag atttttgctg gataaagctt agttgagtgg 83880 aaaccttacc atttgccctt ctagaacctc tgctttaatc tgtagttttc cttttttttt 83940 ttttttttaa atggagatgg gggtctcgct atgttgccca agctggtctc aaactcccaa 84000 gttcaaacta ttctctcacc acagcgttct aaagtgctag gattacagac gtgagccacc 84060 atgcccagcg ctagaactgg gcgctgtacc gaactgaata gaactgctag tctgtagttc 84120 tattcactct cttcacctct gcattggatt tactttgtag agtagcactg tccagtagaa 84180 ctttctgaga tgattacagt tttgtgtatc tgtgctgtcc agtcaagaag ctactggcca 84240 cttgtggcta tcaagtactt gaactgagga aatgattttt acctttttta tttctattta 84300 ttttttatta tttttattta tttatttatt tatttatttt tcagatggaa tctggctctg 84360 tcgcccaggc tggaatgcag tggcatgatc ttggctcact gcaacctcca cctcccaggt 84420 cccagcagtt ctcctgcctc agcctcctga gtagctggga ttacaggcat gcacaaccac 84480 acccggctaa tttttgtatt tttaatagag acagggtttc accatattgg ctaggctggt 84540 cttgaacttg ggagctcaag caatccactc accttggcct ccaaaagtga tgggattaca 84600 ggtgtgagcc accgcgcctg gccttattta tttgtttatt ttttgagatg gaattttgct 84660 cttgttaccc aggctggagt gcaatggcat gatctccgct cactgcaacc tcagcctccg 84720 aagtagctgg gactataggc acacaccacc acacctggcc aatattgtat ttttagtaga 84780 gacgggattt caccatgttg gccaggctgg tctagaactc ctgacctcag gtgatccacc 84840 caccttggcc tcccaaagtg ctggattaca cgtgtgagtc tgtaattggc ctggctgatt 84900 tttacccttt taaaattttc ctggccggac atggtggctc atgcctgtaa ttccagcacc 84960 ttgggaggcc gaggcgtgtg gatcactagg tcaggagttc gagactagcc tgaccaacgt 85020 ggtgaaaccc catctctact aaaaataaaa aaattacctg ggcatggtgg tgtgtgcctg 85080 taattccagc tactcaggag gctgaggtag gagaatcgct tgaacctggg aggcagaggt 85140 tgcagtgagc caagatcgtg tgactgcact ccagcctagt gacagagtga gagtccatct 85200 caaaaaaaaa aaaaaaaatt tgtacttaag tgtaatatat agccagttgt ggccagtaac 85260 tctcataata gacaggacag ccctggagaa tatatggaac ctcagttttg ccttaattgg 85320 tttaaatgtt gtactgtgtc attctcatcc atactcttgc tgttagtgtg gttaagccac 85380 tctccctttt aaacttcacc tagaagccag tcctttatga gacataagac tcccagtggg 85440 taggcttacc aggctctaca gttgctctgc cttcaaggca gcctgtgtgt cagagtcctg 85500 aggctagcgt gtgtgtttac ttgcccataa ctataaatgt taacattgca gatatttcct 85560 ttaattctac tattccagga ttcaggaact aaatctattt tttttttttt ttttgagatg 85620 gagtttcact cttgtcaccc aggctggagt gcaatagcgc gatctcggct cactgcaacc 85680 tccgcctcct gggttctagc aattctcctg cctcagcctc ccgagtagct gggactacag 85740 gcatgcatca ccatgcccag ctaatttttt tatttttagt agagacgggg tttcaccaca 85800 tttggccagg ctggtcccga actcctgaac tcaggtgatc catccacctc agcctcccaa 85860 agtgctcgga ttacaggcat gagtcactgc gctcggcctc attttatttc tcttctctgg 85920 acattttcca tcttaatact ttcatcttga gatttgacac ttaggactgc acagggcatt 85980 ttaggtgtgg acagctttga ttttgtacta aaaatgatta cacttggctt gatttccatt 86040 tctcttcctt ctgttgcttg cctttctaca tggtttagac tgaagcaaca gtgtactaat 86100 atcactagta aaccatctac agtgcctgct attctagggt gtaactgaga gctcagaact 86160 cattctcaca tcaggatttg tgctcctagc atttgtaggt gacgtgttga tctaggtctg 86220 ccttttttgt gttcatgtta gtcactaaca aggggggttt ctagtgctta tggaccacat 86280 ggcaattaaa tgcccatata ttgccctgaa accatctggt ttaacttgtc cctcaatcct 86340 agaagaaacc tgacaatggg attttcacat ttctctaagg aatatgttag taagttattc 86400 cttaagcaca tagattccat ccagattatt agtgcctttt tccccccagc tcaattttag 86460 agataaaaag agctttagac atggtattgt acactcacca aacctctgtt ttatccctaa 86520 atagaccaag agagattacc aagtctccca aggttacata gtaaatgaga aacacctggg 86580 gcagagctca ggcctgctgg tccaagccct gtgttttttc tccactgaag cacctcattg 86640 gagtagcttt ttcataatcc tcatctttct tggaatttct gaaggcaggc aagcctgtat 86700 agccgggcta actttcacct catcctcctg cctcgtctca cctctccctt cccttttcct 86760 tgcctgtgga tgctgacctc tgcatgggtg cctgcttctc taaagctacg atgtaatctg 86820 tagctgtcct tcctcgtttg agacccacat gtgaacaccc catatgtgtg cactttttct 86880 gcgaatggtg atcatctgat tttcaagggg ttagcttgtt ttttaaaaaa tgaaaaaaaa 86940 aatcactgtt tactaatcaa tgagtggtat tggggcaatt tggtagttct cagaaaaaaa 87000 attaatttgg atcaatttct cacatcttat acaaggatga attctaattg gagttaagat 87060 acaaatatag ggctgggcgc agtggctcat gcctgtaatc ccagcacttt gggagtccga 87120 ggcaggtgga tcacgaggtc aggagatcga gaccatcctg gctaacatgg tgaaaccctg 87180 tctctactaa aaaatagaaa aaattagcca ggcgtggtgg tgggcaccta tagtcccagc 87240 tactcaggag gctgaggcag gagaatggat gaacccggga ggcggagctt gcagtgagcc 87300 gagatcgagc cactgcactc cagcctgggc gacagagcga gactccgtct caaaaaaaaa 87360 aaaaaaaaag atgtaaataa aactccagaa atattgaaag aaaccttggg attatttgct 87420 ttgtaacttg gactggtgaa ggtgtttctg tcaccccaaa caccaaaccc atgaaataaa 87480 agacttacaa atttgattac ataaaaatga aatttctggc cggtcgcata atcccagcac 87540 tttgggaggc cgaggtgggc ggatcacgag gtcaggagat cgagaacatc ctggctaaca 87600 cggtgaaacc ctgtgtctac taaaaataca aaaacaaaat cagctgagct tggtgtcaag 87660 tgcctgtagt cctagctgct caggaggctg aggcaggaga atggcgtgaa cctgggaggt 87720 ggagcttgca gtgagccaag atcatgccac tgcactccag cctgggccaa cagagcgagg 87780 ctccatctca aaaaaaaaaa aaaaaaaaat gaaatttctg tgtgacacat ccataccatg 87840 gactactact cacaataaaa aggaacaggc tgggcgcggt ggctcacgcc tgtaatccca 87900 acactttgag aggccgaggc aggcagatca cgaggtcaag agatcgagac catctggcca 87960 acatggtgaa accccgtctc tactaaaatt acaaaaaatt agccgggcat ggtggcaggc 88020 gcctgtagtc ccagctactt gtgaggctga ggcaggagaa tcgcttgaac ctgggaggtg 88080 gaggttgcag tgagccaaga ttgagccact gcactccaca gcctggccac agagcgagac 88140 tccgtctcga aaaaaaaaaa aaggaacaaa ctatgatgtg cacaactcag atggatctca 88200 agggaattat actgcatgaa agtacacaaa aggttacatt ccatggatgc ccattcatat 88260 aacattcttg aaatgacaga attctagaga tggcgagcag gttagcggtt gcaggggttt 88320 agcaaaaggg agagtaagag ggaagtggct gtggctatga aagggtagtc tacagggtct 88380 ttgagatgga aatgttctga atcttgactg atggtgacca catgaatctg aacatgtaat 88440 aaaagagtat agaatgagat acacaaacat acaaattagt gcaagtaaaa ctggggaaat 88500 ctgaacgagg tcagtggatt agatcaaggt ctgtttctct tcagaatctc aaatcttaaa 88560 aggttttgta ttttctaaac acaaataaaa gctggaaagc caatcagtag taatctgaaa 88620 gttcacaatg tcagtagacg tctcatggta ttgggaagaa agataagcat tgaggctaat 88680 atcaccctat gcttgattaa gtttttgttt tatatgttaa aatcactaaa ccgaaagctt 88740 agttgttgtt ctcccacaaa taaaattaag ccctcattta cttttaattc agttccaaat 88800 aggtttgcca tttagatttt tgtccaagaa ctaactcctc cccactcccg gtttttgttt 88860 gtttgttttt taaatttatt tattgagaca ggttctcact ctgtcaccca agccacatgt 88920 agcctcaaac tcttgagctg aagccatcct cccacctcag tctcccaaag tgttggaatt 88980 acaagcatga actaccacac acctcccacc tttaatatat ctaaaatata aattctcttc 89040 tgccttagaa aaaacaggct gggcgcagta gctcatgcct gtaatcacag cactttggga 89100 ggccaaggtg ggtggatcac gaggtcagga gatcgagacc atcctggcta acactgtgaa 89160 accccgtctc tactaaaaaa aaaaaaatat atacaaaaaa aaaaaattag ttggcatgcg 89220 cctgtagttt cagctactcc ggaggctgag gcgggagaat cccttgaacc cagtaggcag 89280 tggttacagt gagccgcgat tgcgccactg cactccagcc tgggcgagag agctagactc 89340 cgtctcgaaa aagaaaaaac aagcatttga ctctgtcttt taagattacc actggattat 89400 catatctgag gcatatacta gttatcatgt tcatttccaa gattctttgc cctctgaatc 89460 tcctgtgctg ctattggagt ctctatatgg tttactacag gacattagtc tgtcatccac 89520 atgtccctag gcacatgtcc ctggacacac tcctcctgga gtggcaggaa tgactagtga 89580 cctccaccga cttcctccct gtcgcttgca tgattcctct gcttctacac cttcccctgt 89640 ctactcaaac tccggtttat ctagaagatt atgagtgctt ttaaaaagat ccaagataaa 89700 acttcaaaag aagtggccca taaaatctat acttttctct tccaaaaggt gatggcatct 89760 ctcctacaaa agaggatgta attcactcag atgtacaaga tgaactggtt cattctgctt 89820 gttacgtatg catctaatta gtttgaatct atgcagtacc accaccttaa gatgtttcca 89880 aaggacaact ctaaactctt actattaaaa aaaaaatgtt ttaagtagaa aggagtatta 89940 agtgaaattt caatattgaa ttcattgcat aaggcaaaca ttagatataa gtgggaaaca 90000 tcttagagga ttttactgtt ttacattttt taggtgaata gcaaccttag atcatcttac 90060 aaaatatggt tactgtcaca aaataaaact tgaaactata ttcagtcatt taaaaaatga 90120 actctttatt ttagctggac cagctattcc ttctgttgtg gcgtatccga aaaggagtca 90180 gaccagcact gcagatagtg acctcaaaga agatggaatt tcctcccgga aatcaagtgg 90240 cagtgctgtt ggaggaaagg gaattgctcc agccagtccc atgcttggga atgcaagtaa 90300 tcctaataag gcggatattc ctgaacgcaa gaaaagctcc actgtcccta gtgtaagtgt 90360 tgttgaacta tagagtggtc ttagggtggt agggttggaa ccagctagac accaggtgtt 90420 cattttactc ctgtggtctc tcgtactgga atgccctctc tactaggcag ccatgcccaa 90480 tcttaactta cagaaatcct acctgctgca ttgtaggtat ttattgttga cactctctta 90540 gttcattctt gctgctgtaa caaaatacct gagactggct aatttataaa gaaaagacat 90600 ttatttctta acaattctgg aggctgagaa gtccaagatg aaggcaccaa caagttccgt 90660 gtctggcaag ggccctgttc tctgcttcca acatgatgcc ttggtgctgg catcctccag 90720 aggagacaaa tgctgtgttg tcatgtggct gaagggacag aaggggtgaa ctcaccccct 90780 caagcccttc tataaaacac taatctggcc gggtgcggtg gctcacgcct gtaatctcag 90840 cactttggga ggccgaggtg ggtagatcac gaggtcagga gttcaagacc agcctggcca 90900 agatggtgaa acgccatctc tactaaaaat acaaaaatta gctgggcgtg gtggcaggca 90960 cctgtaatcc cagctgctca ggaggctgag gtagagaatt gcttgaactc aggaggcaga 91020 ggttacagtg agccgagatc gcatcactgc actctagcct ggcgacagag cgagactcct 91080 tctcaaaaaa aaaaaaaaag atactaatcc cttcatgaag gcggagccct cgtggcctaa 91140 acacctaaaa gactcatctc caaatactgt tttctcctat gggagataaa gcttaaacat 91200 gagttttgga ggggacacat tcagagcata gcacacccac catagacact agatggtgat 91260 aggtccttta tttcaaggcc tggttccagt tatttcggac acctgctgag ccttaggttg 91320 tcaggtgctg agacatggaa agacctcaca gtctaacagg aagtgaatga aaacagtgac 91380 agtgcagatg gaagcctacg tgcacacacg gggaggtctg actgccagtg ccccaaagat 91440 gtggtgttca cactgagtct tcaaagagct ctaatgatga agagtgttgg ctgagttctt 91500 accatgtgcc aaggaccatg ctcagtgctt tttcatggat tagctacatt ttaaggtgaa 91560 aaaaaagttg ggtaattgcc taaaattaca ctgccaacct gaatccagct cttaatccct 91620 atctgtaaca aaatctcccc agtaagtaga taatatttac taaattaaat tgaatccatt 91680 cagcttcaga aacttttttc tgtgtattga agtatgttct atctaaccta acatattaaa 91740 gaatttgcag agaagtaaac aatcttaaga ttattctgta agcacgtttt tccctacaaa 91800 attaacaatg atgcagccct gcctccaact ccagaaaaac cctggactga ggaggctgtg 91860 gagaaaccct gttgctctct gaaagccctt cattgttcca ttctggggac tgggtgcatg 91920 aaggacccca aggggctttg catgtttcat cctctgacct tatgcttagt cgaagtaaac 91980 cctgtgtgaa aagtttattt tggcctccaa atgccataca gtaggattat tgtttccata 92040 ctcctgcagt taaaattccg tctccaacgt ttgttgacag ttaccagaga agatgagggc 92100 attattccct gccttcatga cttctcttcc cctttgctct tgtctacaca cgctgccctt 92160 ctgaaagtga atcatgagta ttttcagtcc acccatacta aattacttac aaaggaaaaa 92220 aggtagttag caacattatt attttctccc atgagtcttt gggatttctc tgtagatggt 92280 ctgctcaagc tgtagtaaaa gtgactgctc tatctttgtt taaaaattat ctgataaaat 92340 tccatagcta tggccaggca cggtggctca cacccgtaat ccctgcactg tgggaggcca 92400 aagtgggcgg atcacttgag gtcaggagtt caagaacagc ctggccaaca tggtgaaatc 92460 ccatctctac taaaaataca aaaattagcc gggtatagtg gcgtgctcct gtaatcctag 92520 ctattcggga ggctgaggca ggagaattgc tttaaccgag aagatggagg ttgcagtgag 92580 ctgagattgt gtcattgcac tccagcctgg gtaacagagc gagactccat ctcaaaaaat 92640 aaaaaaaaaa gtccatagct gtgacttgtt ccttgctttt tagttttttg ttttggaagt 92700 taaattcatg tgaatccaca tgacaatgat taaccatttt aaagtgggca atgcggtggc 92760 atttggtaca ctcacagtat tgtgcagcca acacctgtct ttagtttcag aactttttca 92820 gctccccaga aggagcatct gtttaggctg ggcacagtgg ctaaccccta taatctcagc 92880 gctttgggag gctgaggtgg gaggatcact tgagcccaag agtctgagat cagcctgggc 92940 accagagtga gacctcatct ctacaaaaaa aattaaaaag aaaatagctg agcgtgatga 93000 tgcacacctg tagtaccagc tacttgagag cttgagaggc tcaggtggga agatcatttc 93060 agcccaagag gttgaggctg cagtgagtca tggtcacatc accatattcc agcctgggca 93120 acagagcaag accctgtcct taaaaaaaaa aaaagtggcc gggcgcagtg gctcacgcct 93180 gtaatcccag cactttggga ggctgaagtg ggcagatcac aaggtcaggc gttcaagacc 93240 agcctggcca tcatggtgaa accccatctc tactaaaaat acaaaaatta gccaggcatg 93300 gtggcgggca cctgtaatcc cagctactcg ggaggctaag gcaggagaat cgattgaacc 93360 caggacgcag aagttgcagt gagccgagat ggtgccactg cactccagcc tgggcgaaaa 93420 agcgaggctc catctcaaaa aaaaaaaaaa aaagaacacc tgtatccctt aagtaatcac 93480 tttccattcc tcctctcccc tctgcccccc tcccttcccc ctcattccct ggcaaccacc 93540 agtctgtgtt ctgtctctgg atttgcctgt tctgaatatt ttatataaat ggaaccattt 93600 cacatgtatc cttttgtgtc tacttctttc tttgactcag cataatgttt tcatggttca 93660 tccaatttgt agcatgtatt ttcttttccc tttttttttt tttgagatgg catttcgctc 93720 ctgttgccca gagtgcaatg gcacgatctt ggctcactgc aaactccacc tcccgggttc 93780 aagcgattct cctgcctcag cctcctgagt agctgggatt acaggcgtgc gccaccatgc 93840 ctggataatt ttgtattttt agtagagacg gggtttctcc atgttggtca gtctggtctc 93900 gaactcccga cctcaggtga tccacccgcc tcggcctccc aaagcgctgg gattacaggc 93960 gtgagccact gtgcccagcc tgcagcatgt attagtactt tgtttctttt gggggtaata 94020 ttccattctg tatatatact acaatttaac tgttcatctg ttgatggaca tttatattat 94080 ttccatgtat tggttaaaat gaataatgct actataaaca attgtgtaca aatttctatg 94140 tggacttaaa tgttttaatt tctttccttc tttcttgggg aggagacggt ctcactctgt 94200 cacctaggct aaagtacaat ggtgcaaaca cggctcattg caacttcgac ctcccagacc 94260 caagtgatcc tcccacctca gcctcccaaa tagctgatac cacaggcatg catcaccatg 94320 cctggcttat tttttctgtt tgtttgttgt ggtagagatg aggtctccct atgtagccca 94380 gccaggctgg tctcaaactc ctaggctcaa gcagacatcc tgcctcagcc tcccaaagtg 94440 ctgggattat aggtgttagc ctctgcacct ggctgtgttt tcatttttcc tgggtatata 94500 tttaggaata aaattgttga gtaatatggt aactccatgt ttaatttttt gagaaactgc 94560 caaactgttt tcctcacaac tgtaccattt tatccagcaa tagtgagagt ccctgttttc 94620 ttcacattct ttctaacaat taataattat tttattattt aaagctttcc tagcaggtat 94680 gaagtagtac ctcatgcttt tgatttgcat ttctttaatg accaatgata ttgggcatct 94740 tttcatgtgt ttcttggcca tacgtatagc tttttttttt tttttttttt tgagactgag 94800 ttttgctctt gttacacagg ctggagtgca atggtgcgat ctcagctcac cgcaacctct 94860 acctcccagg tttaagtgat tctcctacct cagcctcctg gcctcctgag tagctgggat 94920 tacaggcatg caccaccaca gccagctaat tttgtatttt tagcagagac gggttttctc 94980 catgttggtc aggctggtct cgaactcctc acctcaggag atccgcccac ctctgcctcc 95040 caaagtattg ggattacagg tgtgagctac cgcgcccagc cgttgtatag cttttttaaa 95100 gaaatatcta ttgaaggtgc cgattttaaa attaggttgt caggccgggc acctcaggag 95160 gctgaggcag gagaatcgct tgaacccagg agggggaggt tgcagtgagc cgagatcgca 95220 ccgctgcatt ccagcatggc gacagagcaa cactgcatct caaaaaagaa aaaaaaaatt 95280 agccaaatgt ggtggcacat gcctgtattc ccagctactt gggaggctgt ggtgggagga 95340 ttgcttgatc ctgggaggca gagccaagat tgctccactg cactccagcc tgggtgacag 95400 agtaagacct tgcctcaaaa aacaagtaaa taaaagactg acattcctat ttatgtagat 95460 aagttcattt tttctcattg tactgtattt accaaataaa tttaagacaa aaatgccctt 95520 ttttatagag taacacagca tctggtggaa tgacacgacg aaatacttat gtttgcagtg 95580 agagaactac agctgataga cactcagtga ttcagaatgg caaagaaaac aggtaggaga 95640 ttctacctgt ttgtaagaaa agttgttttt cccaagagaa atggaagcat gttatttact 95700 gctttgttct tataatcatt ttcaaattaa aactgtaagt attctctgaa ggtaagttaa 95760 atttgtatac taatttttag caagatacta gaattctaaa ttctttttgt tgtccagagc 95820 atcatcctcc tgcctggctg tgtatgatga ttaaaaggct tgagttaaac ccagggggtt 95880 tcctagagga agtagatagt aagctgtggt tatgttgtag aacagtgaat aaggaaatgg 95940 agattgtctt gtgctcatta ataacttgaa caaaagccga aagtggggga agataaggac 96000 gtgaggcaga gagtcatacc tcaggagaat gtacagcacc aggtagcatt aaaggtccgc 96060 agtttaccaa agggtccaga tggctgagtc taaatagaaa cactggagaa gccaagagaa 96120 gtggaaagtc cagccttctg aatgctttta atttggctac atagcaagca tacataggat 96180 gagcgtgact ttaaaaagac aatgatgtta catgtcaata aggagagcat cctatcatgc 96240 acatgatttt cttggggttt tgaaataaaa taagtagagt cagtaaaatg ggattggata 96300 ggtgtagaga ctctgaggtg gggttagtct gggtggagag aaaagagaag ttagattggt 96360 agagaatagc taaatatgct atattctgtt cagaacaaat ttcccaggct ggcatggatt 96420 ttccttcttt gtttttggaa gagcagtaag tgtacatcat tcttttaact tgaacattgt 96480 ttgcatcaga attgttctga catttacttt ctgcagttac ggtgtgaggt tctgtttctt 96540 aatcctaacc tatgtttgat ctcattagtc aaacaaaatg tttgatcact acattttgtg 96600 acagcctaat caaaaattct ctcctccttc atccttagtt ccaaactgct tatcacttcc 96660 ctgttccata ggccacttag gattgagttg atatgaggag taacatttgt tctgtaagat 96720 tatggttatt attcgttgaa tttttagcaa tatttacatt ctcttgttct cttttatgac 96780 tcgtgtgtgt gtgtgtgtgt gtttcaacat tgtcttttct tatgattaaa aactactaag 96840 aacttgagta tagcagctgg ggtagatagt ttacagataa tgattgatat aggtatttct 96900 tttttttttt tttttttttt ttttttgaga cagagtctca ctctgtcacc caggctggag 96960 tgcagtggcc tgatctcggc tcactgcaag ctccgcctcc cgggttcacg ccattctcct 97020 gcctcagcct cccgagtagc tgggactaca ggcgcctgcc accactccca ggtaattttt 97080 tgtattttta gtagagacgg ggtttcacca tgttagccag gatggtctcg atctcctgac 97140 cttgtgatcc acccacctcg gcctcccaaa gtgctgggat tacaggcgtg agccaccgta 97200 cccagccagg tatttcttaa tagtagtcta ctctctggta gatttgtatt atagttcagt 97260 tcttaacttt caaatttctt ctagttcaaa agtgacttaa tagaaaatat caatagaaga 97320 cttaattaga agtttctggt gttacagatt gtagtctaca ggactacaga aatgtttcat 97380 ggtcaggcac agtggctcac acctgtaatc ccagcactct gggaggccaa ggcaggtgga 97440 tcacttgagg tcaggagttc gtgaccagcc tggccaacat ggtgaaaccc cgtctctact 97500 aaaaatacga aaattaacca tgtgtggtgg cacgcgcctg tagtcccagc taccagggag 97560 actgaggcag gagaattact tgaacccagg aggcagagtt tgcagtgagc tgagatcgcg 97620 ccactgcagt ccagcctgga caacagagca agactcatct aaaaaaaaaa aaagttgttt 97680 cattaccttt actacttcta aattctgaaa atgcactacc cagctccttc tgaactttag 97740 gatatgtgac tgtctgaggt gcagagtcag tctcctcaag ggtgttaaga ctaagatctg 97800 ctctctgctt ccatcctgtg ctacacgtat catacaccag gcacatgcat atatgcctgt 97860 gtcttctgat tctcttttcc taaaattttt cgtttcactc tttggaaata atatttaaga 97920 cttctttaat ttctttggca atatttcctt tctgcattcc ccctggagac acctgccttg 97980 ttggggccaa ctctttcata tcctgacctg tctcgttgct tctcttagca tcagtcattg 98040 aaatactttg cttctgaagg tgtcgtcttt ctctttagtc aaagagagtc tcaccgccaa 98100 accttaaaag acagggccca ttgttttaat aatatttgga ctttcctttc ctatcttata 98160 aatgaattaa aagactggaa aattattggg tttaacttct ttagttaagg cactaaatgt 98220 gacagctttc ccttaaaatt gatgcttgag tcttcagtga gatacaatta gctttccctg 98280 cacagatgac ttcttcggca aattgttcct tttccctctc ttcacccacc catatatctt 98340 atttcttcac taatttaaaa tatgagtgag ctgtaggatg cctggaccta tgttaacttc 98400 tgaaacaaac atgttaagtt ccaattttag ccttaaatat tttcgacatt tagccttaaa 98460 tgttttcttc tttatatgag tgttcatacg gaatttcaac atgagaataa tgatggctga 98520 ttttttaaaa actgtgtgaa ggtttaaacc agagactcca catatcttaa gcccttacta 98580 ccttcaaaat cctactgtaa acaggatatt ttaaaggctt actaaatgca aggctttttc 98640 tcgtcatagg caatataatt aattttaatt attttttctt ttgaaaatta gttttattaa 98700 ttggttctta ctaggccaat ctttgtttta tcagctttcc tgtgggaaca ataaagttaa 98760 tgacacaggg agcataggtt agggccagtt gttactttgg cacaaacacc tgtgattcgt 98820 tttctgagac tagttttaaa ctggtcgtgc tgaaggccag cccactttct gctgtaatta 98880 ttctgccttc tgatgttatt ctcgaaaaat ggccagattc caggcgtgac taatggttgg 98940 tgtaaactag gcatgagaag gtttgattac accttcaatt gtatgattct atcatgatgg 99000 tataggtttg cagtgagtct taaaccacat agcactgaca cgtagtgagt gctcaaacaa 99060 atgttagctt tcattattaa ttatagactg ttcagacaag gttatgcaga attttcttag 99120 tttgcactga ctaaacctaa tctttcaagt taataatgtt gctaaaccac ctacatagca 99180 gagacactct gctcctacga tttcatatgc tttagatacc cggacgacac tgtttagact 99240 acttcattta aagaccccag agaagtgacg tgctggtgtt acagtaatgc aaacagaatc 99300 ttttcttttt tttttttctt tttttctttt tttttctgcg atggagtctc gctcttttac 99360 ccaggctaga gtgcaatggc acgatctcgg gtcactgcaa cctccatctc ccgggttcat 99420 gcagttcttc tgcctcagcc tcccagtagc tgggattaca ggcacacacc accacacctg 99480 gctgattttt gtatttttaa tagaaatggg gtttcactag gccaggctgg tctcaaattc 99540 ctgacttcaa gtgatccacc tgccttggcc tcccaaagtg ctgtgattac aggcatgagc 99600 cactgcacca ggcccagaat ctttcaaata aattttagat attggatatt actgtaaagt 99660 ttcaaaattg tgaagtggct tattatttta tccactttac cctgcatcaa gtcacataga 99720 gattgagcag agaaggattg aggacactta gcgtatgtat ctttggacta catataaaag 99780 ttgctttttt taggctgggc gcggtggctc ccacctgtaa tcccagcatt ttaggaggct 99840 gaggcaggtg gatcatgagg tcaggagatc aagaccatcc tgaccaacat ggtgaaaccc 99900 tgtctaaaat taattagctg ggcgtggtgg catgcgcctg tagtcccagc tacttgggaa 99960 gctgaggcag gagaatcact tgaacccagg aggcagaggt tgcagtgagc cgagatagcg 100020 ccactgcact ccagcctggc aacagagcaa gactctgtct caaaaacaaa aaaaaagggc 100080 tgagcacagt ggcttacgcc tgtaatccca gcactttgcg aagctgaggt gggcggatca 100140 cctgaggtca ggagtttgag accagcctga ccaacatgga gaaaccccat ctctactaaa 100200 aatacaaaaa aaaaaaaaaa aattagccag gcatggtggc gcatgcctat aatcccagct 100260 acttgggagg ccgaggcagg agaatcgctt gaacccagga ggcagaggtt gcggtgagcc 100320 aagattgcac cattgcactc cagcctgggc aacaagagca aaaccccatc tcaaagaaaa 100380 aaaaaaaagg ctgctttttt tttttttttt ttttttttaa gatggatgga gtcttgcagt 100440 gtggcccagg ctgcagtaca gtggctagtc acagacatga tcatagtgta ctgtagcctt 100500 gaactcctgg gctcaaatga tcctcctgtg ttggcttctt gagtagctgg gactacaggc 100560 atggaccacc acacctggct aattttttat tttttgtaga gatgaagtct tcccatgtta 100620 cccaggctgg tctcaaattc ctggcctcaa gggatcctcc agtctcagcc ttccaaaaca 100680 ctaggtttac aggcatgagc caccatgcct gtccttggac tatattttta attctgcttt 100740 tgcccagtca cttgagtatg cttttaatca gcagccaata catttcttat cagaatgttc 100800 tgataggggc tagaggtcat agcaacaaat tcaaagtgcc tatcctttag taaccttaag 100860 tggattaatg ttttagaaaa gatagtcaag actgggcaca gtggctcaca agccctgtca 100920 tcccagcact tcgggaggcc tagacaggaa gatcacttga ggccaagagt tcaaggtcag 100980 cctggacaaa gtagcctgag acccccgtct ctataaaaat atatatattt tttaatttta 101040 aaagatagtc atgaatacaa aacagttggg aagattaata ggaactctct tcatgcaact 101100 tgaattttaa gaaaaacatt gctatttcta tcaattaagg tttaaatgta gaccaggcat 101160 gatggctcac gccaccatgt aatcccagca ctttgggagg ccatgacagg aggattgctt 101220 gaacccagga gttcaagacc agcctgggca acatggtaaa attccatctc taccaaaaat 101280 acaaaaaaat tagctgggtg ccaggcacag tgcctcacac ctataatccc agcactttgg 101340 gaggccgagg caagtgcatc acctggggtc gggagttcaa gaccagcctg gccaacatgg 101400 tgaaacccca tctctactaa aaatacaaaa attagctagg tgtggtggta catgccggta 101460 gtcccagcta tttgggaggc tgaggcagga ggatggtttg agcctgggag atggaggttg 101520 tagtgagccg agatcacacc attgtattcc agcctggacg gtagagccag acccagtctc 101580 aaaaaaataa aaataaaaaa attaatgtaa tacagctact atgaacccac aaaaacttta 101640 aaaaaatttt ttaattatta aaaaaatgta atgctgctac tgttttgctt atgtagattg 101700 tgggctagaa taggaaataa aactatcatt tttaacactt ttctgggggg aaatattatt 101760 cgaatttcat aacaattagc ttgatgacct tttagaatat atatgtagac cattgtgatg 101820 ttggggctgc ctgttgtaaa taggattttt gtgagcttga ttgtacaaaa ttgaaatttg 101880 caaacatctt actgagttta ctgtttcctt ctgggtgtta ggcctactgt agaaatcaga 101940 aaaatcggaa atattttgtt gcttgctttg tgaaactcat gatgtgacat ggattaaatt 102000 tctaaaaagt ttatttaatt gcctttaaaa ttagtatgtc aaaaagttta tttatttatt 102060 tatttattta ttttaatttg aaacagtctc tctctgtcac ccagactgga gtttagtggc 102120 atgatctcag ctcactgcag ctaccacccc cgggtttaaa cgattcttat acctcagcct 102180 cctgagtagc taggattaca ggggcccgcc accacaccca actaattttt gtatttttag 102240 tagtgatggg tttttgccat gttggccagg ctggtctgga actcctggcc tcaagcaatc 102300 cacccgcctc agcctcccaa agtgctggga ttacaggcgt gggccaccac gcccagcctc 102360 aagttaataa tttataatcc cagcactttg ggaggccaag acgggcggat tgcctaagct 102420 caggagttcc agaccagcct gggccatgac gaaaccctat ctctacaaaa tttttttttt 102480 taatttataa tgagaaaata aatttacatt tccttcttag gtctctagag gatccatttt 102540 ttttctgcaa agcatctgtc cacaccctct taccatgctt gtatgcctta aagatctagc 102600 ttggcctgtc agcagtgtgc ttcattggga atcgatgcag caccctcctg cctgcaagct 102660 gactaaaagc cttttccttc tccaaagact ttgggaccat ttgtattcac cagggaaagg 102720 gtcaaacaac tcctgcatct tcttcccctg cttttcttgg cacatctact gatactagct 102780 cctaatttgg gcaagaaaaa agtcaacaac tggaggtaga gtgtgttgac cctggactca 102840 ccctgaaagg taagggcaca agagatagtt gtatttagct gtatcttgtt agaaaaatac 102900 atttgtgtag ccaggcgcgg tggctcacac ctgtaatccc agcactttgg gaggctgagc 102960 cgggtggatc acgaggtcag gagttcaaga ccaccctggc taacatggtg aaatcccgtc 103020 tctacgaaaa atacaaaaaa ttagccgggc gtggtggtgg gtgcctgtag tcccagctac 103080 ttgggagact gaggcaggag aatggcgtga acccaggagg cagagcttgc agtgagcaga 103140 gatcacactg cactccagcc tgggcgacag agtgagactc cgtctcaaaa aaaaaaaaat 103200 aaataacatt tgtgtgcatt catgtgtacg tgtgtctgta cacatgtaca agaaacaacc 103260 gagcatttct ttaaaaacct tagtcagtta gaactgtctc tgttgcaggt gacagaaaac 103320 ccagtctaaa caggcttaaa tataaaaggg ctgttagttc atataactaa aaaaagtcca 103380 gggctagagt tacggtccat ctgcttctgc ctccttggcc gtagttccat gattgggctc 103440 ggcaccatca gacataactt cttcccacac acagctgtgc tcaagcagaa gccctcaggt 103500 ggtctccttg atctgtgtgg ggcctagagt tacttccacc acagcaaaat ctcattattg 103560 ttgctgcagg aaaagaatgg atactgagtg gcagaaatac gaaactggat aaatttttgc 103620 ctttataaga catttgtttc cagtccttct cagactgtat aaattctccc aagtcagaga 103680 gcagaattct caaacaaaaa tgcagggaca gaaccaggca cagtggctca caaggcctat 103740 aatcccagca ctttgggagg ctgaggcaga aggatcagtt gaacccagga gttcaagacc 103800 agcctagcca tcatagtgag acaccatctc tacaaaaaat ttaaaaatta gccaggcata 103860 gtggcacact cctgtggctg agataggagg atcgcttgac cccaggaggt tgaggctgca 103920 gcaagctgtg atagtgccac tgcactctag cctgggtgaa tacagcaaga ccctgtctcc 103980 aaaaataaaa atttaaataa aaatgtttta attcagggac aagcagttct tgattttaaa 104040 gacgtatctg ctttatgttg gggctatcag aattccagtt gctaattgat taatctgctc 104100 tgttaggaga acgtttgttg aatacctgcc ctgccaggct ttctgcttac actggtgggg 104160 ggcacattga caagattttg ggctgtgttt tcagggacct ccagcagaga agggtcacta 104220 ggatacaatg tggcatgcat gagctaaaaa cagaagtgat ttaactctca gagggaaaat 104280 ggatcagata acacttcata gagatgacga catctggttc atttttgaag agtgaataga 104340 aagatcccag ccctccatac caagagaata actcctgcaa acattttctg acctggtgcc 104400 tgaccaatgg accacacatc atcaggaagg ctagaaagtt gagttccagg agggaatgat 104460 agaaggcaaa gcctagagag atggcagggg ccagacttca ggggcctggg gatgtgtact 104520 tgcactccaa gtcgaggaga ctgaactttt taccaaagac ttggtgttct gtagaagtat 104580 gagcagatga gtgacaagat ctggtttata tgttaagaag ttctcagctg taaggtgaat 104640 agattggagg tgttggggca aaggcagggg acccagtgaa gacaaggagc gaggctctat 104700 actgaggccc tggtagaagt ggagcagatg ggccacctga agagctacta ggaagttgac 104760 accaataaga tgtcgttagt ctgtgtatgg ggagtgaggg aaaggaagaa cttgccagtt 104820 tccaggtttt aatccttcca aaggatttct gcagaaagac actggaagag ctgtattcct 104880 acttatcctt aaaggaaaga aactgccctt gaattccaag gacatgatta ctgagctgga 104940 attccaaata atgtgtccca gtgagactcc tgggggtagt tttgtccggt aactgtgtgt 105000 gttagatgaa actgcagact tccagccact ctatcttacc atgtctgtta acagttaatt 105060 tgctctgatg aatttgccca tcttggagaa gggggctctg ctgactgttt tttatcttgg 105120 agtaaatgtc ctttgattag gaaggttcta gactatattg ttaaaacaac attggaaggc 105180 tgggcatggt ggctcatgcc tgtaatccca acactttggg aggccaaggc aggcgtatcg 105240 tttgagccca ggagttcaag accagcctgg gcaacatgac aaaactctct ctactaaaaa 105300 tacaaaatta gccaggcatg gtggagtgcg cctgtattcc cagccactta ggaggctgag 105360 gtgggagaac cacctgagcc caggaagttg aggctgcatt gagccatgat cgtaccactg 105420 cactgcagct aggtgacagc aaaaccctgt ctcaaaaaaa caccaaacaa cataggatac 105480 tgttatataa cagctagttc ctatacctgt aatcttatta attcatttaa aatacatcag 105540 tgcgggccgg gtgtggtggc tcacacttat aatcccaaca ctttggaagg ccaaggcggg 105600 aggatcactt gaagccacaa atttaagacc agcctggata atatagcgag accctgtctc 105660 tacaaaaaaa ataaataaag aaaaaataaa atatgtcagt gtgttttggg aacaggttga 105720 gagcatagca tgctcatatt atatattcat tttaccactt agatttgaat tgaggcatct 105780 cttagaatag gttttatttt gtgcataccc tcagaggaca ttgtccctct catagaaaca 105840 gatattcaaa catgtaatct gctattttat tttaaggtaa tagattttta tgattactgt 105900 ttggaaaaaa aaaaaagcaa ctataggtta gcagttgctc cagaatctcc aaaacaagct 105960 taggagattt ttattcttta ccagaaatga gatggcttgg gaggaaggtg tgtgcattac 106020 acacaggttg ctctgtggtc actgctcctt catactgtgt ttctgcctct gcctgaaccc 106080 aaacccctcc cagcctgaca gaaatgttcg cttacgcagc tagccctgcc tcagtttgta 106140 catctacctg tcggctgaga catcagaagt cgatgtccat gtcagcctct gggcacccca 106200 agatgatgtt acctccaata gacagtgaag gagataactt caaggctatg taagaaaaat 106260 gcacattctt tgtgaaacta atgtgtaccc actgcttctt aattttgtgc tgtggatttt 106320 atagtctgag attttgttac agttgtccac aaggcgtcct tcctccatgt gattttagtt 106380 aagcacatct tttgtcatga tgtcctcggt tgtgtattgt gtgtcttcgt gccttcgcgt 106440 actacaaaat gtataagaag ttcctgctcc tcccaagttg ctttcagcat gtggaatttt 106500 atacatatat catttgttta cttccaaaac tttttagttg cctgttcttc agttacgcca 106560 gcatatcctt tatttctttc gtattggcac agttctctat gtaagcaatt tgagagggaa 106620 gcaaagggga aaagtttgag ttagctgttc tctgtcctag aatttccctg cattaatctt 106680 gtccttgaaa atatatataa tactggtccc ttaaactcca tgaggctttg tctcattatg 106740 tattgttctt ttggtaccct ttcccactta acttaccttt tgctcctaag tcctataaaa 106800 taccccttgg atctggattt tttatacccg attttctcca ctgtgtataa aaggtatatt 106860 gtgactgtaa atttttgtat atcatgttct gagagcttct tactttctga tctcatagca 106920 ctattcctga tcagagaact ccagttgctt caacacacag tatcagtagt gcagccaccc 106980 cagatcgaat ccgcttccca agaggcactg ccagtcgtag cactttccac ggccagcccc 107040 gggaacggcg aaccgcaaca tataatggcc ctcctgcctc tcccagcctg tcccatgaag 107100 ccacaccatt gtcccagact cgaagccgag gctccactaa tctctttagt aaattaactt 107160 caaaactcac aaggaggtaa gtgctaggtg ctggttgttt tggagtgaac acatagagca 107220 aaaggaaaga tgtctttttt gttgtgtgaa gccactgcta cctggatgct tttcagtctg 107280 ccttcagctc atggttttct ggttttatat atatctaact ttatacttta aaacctttaa 107340 agtagaaatt gtagagctca gagtcttcat aacactaaaa gttaactagc atttaggagg 107400 tttttgttac tgttctttaa ttattgttcc ttttgctcac gtatctgtct gcatggcatg 107460 ttcgttgttg ttgagcaggc agacctcgag ttctggtgtg gggtgggggg ctggcactct 107520 gtaacaatca gcaggaaaga ccatgcatgt ctttcataat catctccagc cccagaggaa 107580 agagacagct tctcttttct cactttattg ttgttaagga gcagtaaatt tcttaagagt 107640 ccaaatcttg aaagtttctc tctcagtctt agtaatttat attgagagca aggtaatgtc 107700 cttaaatggt atagttttgg agataaaatc tctggtttat tatcaaaaaa taatgttact 107760 attattaagt tatttttatt aaatgctgca tgattcttgg ctgatggtcc tggaaatttt 107820 ttaaaatgtt ccatgtgtct agtaggctag catctggggc ctcagtaaac tctcaagggt 107880 cctttgagat taacttcatt tccgtaacct tgttacctgt tacaaaagcc cagaaatact 107940 ggctgcttca agaggaaaaa gaaaggatac cactttgaac cgtaaacaat agtgaaaggc 108000 acctttccga gaaggagccc aggaaaccca agagcccctg cagtttcagg acatccttgg 108060 agaaatgttt tgaaattaag cctagctgtg gatgagtctg tctgtggtat cttgcaggtt 108120 tgcctaggac ggaccctctt atttgtcatc tgtagcttac aggacagcag taggagtctt 108180 tcccgagtct gaaatgatca gtaccctcct ctgtttttct gcattcccta tggcctatag 108240 cctattactt catttcactc catctccatt atttaaacta agctaaatgt aatcttgtat 108300 tcgatcatct gaaacttcgg tgagtcacat taaagtctga tctacttatt attaggacat 108360 ggaatagatt agggagtatt cctttttttt ttttttttaa tttttttttt ttttttgaga 108420 cggagtctca ctgtcgccaa ggctggagtg cagtggtgca atcttggcta acttcaacct 108480 ccgcctagat tagggaggat ctctgacatc cattagcatt aactagaagt cagacgcatc 108540 agaaggtttc tatttaccct cttactcact tcttagcccc tttattaagc cacctcattt 108600 taaactgggc aaaacagaat cagccaaaga gctttgccag gtttgggaca cccattcatg 108660 accagccagc aagagagaca gtgagttcaa tctgagaaga gtctggtgac tcccagaata 108720 aaccattcct tcctggccag ccgtggtggt tcatgcttgt aatcccagca ctttgggagg 108780 ccaaggcggg tggatctcct gaagtcagga gtttgagacc agcctgacca atatggtgaa 108840 accccatctc tactaaaaat acaaaaatta gccaggcatg gtggtgggcg cctggagtcc 108900 cagctaccca ggaggctgag acaggagaat tgcttgaacc cggaaggcag aggttgcagt 108960 gagccgagat cgtaccactg cactctagtc tgggtgacaa agtgagactg tgtctcaaaa 109020 aaaaaaaaaa aaaaactttg agtttatttt tcttctttgt actctttttt cagtcatttt 109080 aagactagta ttgggctggg ccggatggct catgcctgta atctcagcac tttgggaggc 109140 caagaggggc ggatcacctg gggtcaggag tttgagacag gcctggccaa catggcaaaa 109200 ccccatctct actaaaaata caaaaattag ccaggggtgg tggcgctcgc ctgtagtccc 109260 agctactctg gaggctgagc caggagaatc gcttgaacct gggaggcgga ggttgcatct 109320 agccaagatc gcgccgctgc actccagcct gggtgatgac agagcgagac tccatctcaa 109380 aaaaaaaaaa aaaaaaagac tagcattgat tggccaggca tggtggctca cgcctgtaat 109440 cccagcactt tgggaggccg aggcaggtgg atcacctgag gtcgggaatt tgagaccatc 109500 ctagccaaca tggagaaacc cagtctctac taaaaataca aaattagccg ggcatggtgg 109560 cacatgcctg taatcccagg gagactgagg caggagaatc acttgaacct gggaggcaga 109620 ggttgtggtg agccgaggtc acgccattgc actccagcct gggcaacaac agtgaaactc 109680 tgtctcaaaa aaaaaaaaaa aaaaaaagac tagcattgat tttattacat gtatataatg 109740 gaaaaaagga aacaagatta tcattgataa aaatatcagc tacctctttt tttttcccca 109800 cagagtctca ctttatcgcc caggctggag tgcagtggca tgatctcagc ccatggcaat 109860 cttcgccttc caggttcaag tgattcctgt gcttcagcct cccgagtggc tgggactaca 109920 ggcgcccacc atcgcgcctg gctaattttt gtatttttag tagagacccg gttttatcat 109980 gttggccagg ctggtcttga actgctgacc tcaagtgatc caccgcgccc agccaaagct 110040 accattattt gaatgcttat tatgtgctaa ggtagatgtc tatatacata gatacagtca 110100 tattcatttt atacatataa atatgtatgt acattatata tatgcatatt ttatgtataa 110160 aatgagtatc atatatatac acttatatgt gtgaaatgag tatatttata cacatgtaca 110220 agtgtgcaca cacactcatt taatttaatc tccatagtcc accccatttt gtaaaagaaa 110280 ctgaaggtga agtttggaga gattaaggca catattttaa aaagtatcag ggcctggtgc 110340 agtggcttac gcctgtaatc ccagcacttt gggaggccaa ggcaggtaga tcaggtagat 110400 cactcaaggt caggagttca agaccagcct ggccaacatg gtgaaacccc atctctacta 110460 aaaatacaaa aattagcttg atgttgtggc acatgcttgt aattccagct atttggaagg 110520 gtgaggcaag aggatggctt gaacccagga ggcgtaggtt gcagagagcc aagatcacac 110580 cattgcactc cagcctggcg acagattgac actccatctc aaaaaaaaaa aaaagtatca 110640 ggctgagcca ggtctatctg accagactct gcccttttag ccactacact gtgctccctt 110700 tagaaaaaac agttgcaggc gtgacagttg cattacattt atttcatata tttgaattaa 110760 aattggagac tcaggcttgt tgtttaaaaa aattctaagt ggtcatcttg tttacaattt 110820 ccaggctatt tccacacccc cagatttttg cagaaagata gtcttttatc ctgtcttgtt 110880 aaagtttatt aactgaggtc tgttgtctcc cttagtgcta tacttaatca gctactgaca 110940 gtttgttctt aaaaacaatc aaaatccttg ctagagtggt tgaaagttct tatcctcctt 111000 tagcaataat tggtaggatt aaaattgcat tgcctgaaag gctgaggtgt ttgcacttag 111060 atgctgcaag ggagacctgg tgtggccagg tctggagagg gctgcagtca ggaagcctgc 111120 actctgcttg gagagcacat ggctttggag agctatgggc tatttgtagt cgggagcaaa 111180 actctctctc cttaggatca ggcctggcct tgctcagaga ctgcagagtc acccataccc 111240 tgcaggcaga acagccaggc acacgcaagt gcctctgccc tcaggttgat ccatgttgcc 111300 atgggcagtg tgaaggcctc ctcctgcagc tgcctccttc ctctttatac ctcaagggat 111360 tataggagga aaagttaaga aaagcacttc tatagtaatt gaccagtaac cctgattttt 111420 ccggtggaag attaggaaat gtccaggcac catggctcat gcctataatc ccagcacttt 111480 gggaggctga gccacatgga tcacttgagc ccaggagttc aagaccagcc tggacaacat 111540 aatgagaccc catctctact tttttttttt ttgagacgga gtctcgctct gtcacccagg 111600 ctggagtgca gtggcacgat cttggttcac tgcagtgtct gcctcctggg ttcaagcgat 111660 tatcctgcct cagcctcccc agtagctggg actacaggtg catgtcacca cgcccagcta 111720 atttttgtat ttttagtaga gacagggttt caccatgttg gccaggctgg tcacaaactc 111780 ctgacctcaa gtgatccgcc cacctcagcc tcccaaagtg ctgggattac aggcatgagc 111840 caccacaccc agccttttat ttttttaata aaaagaaaaa aagttgggaa atgggccagg 111900 cactgtacat ggctcatgcc agtaatcctg acactttggg ggaaggtgag gcaggaggat 111960 catttgaagc caggagttca aggctgacct gggcaacata atgagacccc catctctaca 112020 aaaacatttt ttaattagcc aggagtggtg gtgcatgcct gtagtcccag ctacttgaga 112080 ggctgaggca ggaggattgc tcaaacccag gagtttgagg ttacagtgag ctatgatcac 112140 accactgtac tccagcctgg gtgattgagc aagaccttgt ctctaaataa ataaatattt 112200 aaaagtaaaa gaaaattagg aaacgaaaat atatcttaaa gacttttaat cattttcaac 112260 taaaatattt tggataatac tgccttatat ttaggcagta tatattaaag attttcacat 112320 gcatgattgt actgtcgatg accctgctgt atactactga tctgattatg aagtggttgg 112380 cagttattta tagtagttag acttttttta aattggtaga ttaataccta aagcaatgcc 112440 aaagttatcc caaccaagga tttcattaat ttagtcattc agcagatata gatgactgcc 112500 tgctatgtac agttacagaa ctggacttta caaatagaaa ttaacaaact cagcctattt 112560 ttgcccaggg aagctgatcg cattgatcat aaaaacctca ttcgtagttt gaaaaatatt 112620 ttgtcagatt attttcagca caacctgcac atctccttaa atttttttaa tgaccagagt 112680 tacccctttt aaatagaatt cttcgtgttt actacataaa agtaaaattc atactgttgt 112740 cttgacactt agtttttatt cattttgaga tctaagactg aaaagtctac ttgtttaaat 112800 ccccaatcac ctattttttt cttaaaaagt gatttatgtc ataccactgt agcaaataat 112860 atgttaatgt ttgtttcatt ttcctgaaag aagtaatgat ttttacttag tggttaaata 112920 ttttataatg aaatttttaa tgtttatgta ataaggcaag atttttgttc ataaggtatt 112980 ctgaagaaaa ttgaaaaatg ttttattact agtacattgc aaatgtcttg tcacttcttt 113040 aacataagac ataaatttta tcttagaaag tgtatctggc cattgcagtg gctcatgcct 113100 gtaatcccaa cactctggga ggccgaggtg ggctgatcac ttgaggccag gagttcgaga 113160 ctagactggc caacatggca aaaccctatc tctactgaaa atatagaaat taactgggca 113220 tggtggcgcg cgcctgtgat tccagctact caggagactg aggcacaaga attgcttcaa 113280 cccgggaggc ataggttgca ttgagctgag attgcaccac tgcactgtga cagagtgaga 113340 ctctgtcttt aaaaaaaaaa aaaaagtgtg tccatctgaa gaacttttaa ttacagctaa 113400 tgtcaatatg tactgctaaa ccttttctta cttttttttg atatattaaa agatcacata 113460 ctcatttaat tacctgtaat aattttactt tttcaacaaa tgtcagcatt tctttgttca 113520 ttggccagag aaggaaccct gctttgtttt ttttctttct ctctgtaatt gattagattt 113580 tctgtttaat ttgtgcgagt tatttttgtt aatttttttt tttttttact taatttcttt 113640 tagaaacatg tcattcaggt ttatcaaaag gtaggattta tatatacaca tttatttttc 113700 aatcctcact cccaaatggc tcctgcaatt aacattaggt ttggctttct ggccctgttt 113760 tttccttata aactaaactt tctgctgata actaaatact taattccttg aaaggaaatt 113820 gaaaagaaat agattaactc tgtcaggcat tttaaaggga ctatggtacc catgcaacaa 113880 aaggctgatg catgctctgt gtattttctt tctttggtag aattatttag catccaaata 113940 attctgtctt catactaata acacttagca tgctcctgtt tgaagaatga gatgctcagt 114000 aatgttaatg tacaattaat gattatccac aggcatgcaa aaggtaagta ttagttgtgt 114060 tatttttatt tcactgagga tggaattagc aaaaggcttt aaaatgacag gaaaattagc 114120 taatacagaa aacaagcata aaattcaaag ctacagcctc atttgatttg gctttttcag 114180 aaattaaaat gtgaacagct gcgtagcaga aatgttttaa tattttcaga gttgaaagcc 114240 actttccagc aaccactgaa gaaagagtat ctcattattt ttacttaaag cactacagaa 114300 agtggtgttc tgattttatt aatatttttt aggccaggca tggtggctta tgcctgtaat 114360 cccagcactt tgggcggatc acttgagccc aggagttcaa gaccatcctg gacaacatgg 114420 caaaaccccg cctctacaaa taatataaaa attagccggg catggtggca cgcatctgtg 114480 gtcccagcta ctcaggaggc tgaggcagga ggatcacctg agccctggga ggtcaaggtt 114540 gcagtgagcc atgatcatgc cactgtgctc cagcctaggg gagtgagacc ctgcctcaaa 114600 aaagaaaaac atattttttg atggtgataa tcaagaaacc aaaaatattg ctttcttaat 114660 gcacacatga ggcaggaaat ctttcctgaa gggctacatt gtacctgtgc ctctcaagtc 114720 accagaaggc caagctgcag gtcaaaactg cgggaaaagc actttcttcc tgttggcagt 114780 tccattctat tattattttt taattgatct tcccacttgt ctgatttttc cttggacaga 114840 acaggtaata actgaatata gaatccagct gatagcctca ttggctttta attggaaacc 114900 cattatactg tgtggcacaa ttagaaagtg agaataaccc cattctgagg ccgagtgtgc 114960 tcaggctgaa gagccagcag gagtgcccgc tgtgcgtgcg tggtgtgcgg tgtgtgcagt 115020 gtgcagcgtg cagcggtatg gcatgcaatg tgtgtgatgt atgcagtgtg cagcatggag 115080 ctggcccctg tgcacacccc tgcagccttg tggaagaagg tagcgctggc tcagtcaaat 115140 gagaggaaga gttttcataa gcccggctgg tgtttaaaac gtgttttggc tttgttcatt 115200 ttatggtgtt ggtgttggta ttggtggtca tgtactggca tgtaagattt cttttctctt 115260 tccctcttct ctctgcttct acattctgtt cattgaggct tccaactgaa tatgagagga 115320 acgggagata tgagggctca aggtgaggga aatgattttt acttaaaatt tttttcaggt 115380 gtttacttca tttctgtggc agaggcgact attttctgaa tgttccctac tgtattcctg 115440 ctgtctctgt aggagttacg tagagagggt ggccacaagg gggcgccagc ctgccatgag 115500 caccgtccag atccagcttg gcctccgctc tacatttgtg tttgtgttca aagcacggcg 115560 aggccttcag ccttcctgcc tgggcgttaa cagcctttgg aggtctgggt tgggtagcag 115620 tgctcagaga aataagccct cagttcatca ctgacccctc acatggttct cttcacaagg 115680 aataaaagca aagtacttaa tagaatgtcc atatttcaaa ctagatatgt tttcttctaa 115740 gaatggtgat tatatagttg agtgttttgc ttttcttttt tttttttctg agacacggtg 115800 tcgctctgtc acccagtctg gagtgtagtg gcgtgatcag ggcttactcg agttcccagg 115860 cttatgcgat cctcccacct cagcctcctg agtatctgga accacaggca ctgccaccat 115920 gctggctaat ttttgtattt tctgtagaga tgggggtctc gccatgttgg ccgggctggt 115980 ctcaaactcc taggctcaag tgaacctccc acctcaacct cccaaagtgc tgggatgaca 116040 gacatgagcc cccgcaccca gccttgcttt tccattttgt atagttatca ttgcatggat 116100 ttgaggggag gcaatcacca ggtagcctta ggactctatg tcccttgttc taggtctctt 116160 ccacttgtgc ccttttttta tgccagtcac cagcaggcta tttcaggtcc tcattcaccg 116220 tttgtccttt tggtctttct aaaagcccgc ttctggtcat atgacctttc tgaattaaaa 116280 ccttcagtga cctccactat ttcacctggc cacgtccctg cgaggccctg agtggcgtgg 116340 tccaggctgc cccagcagcc ccagctctgc tgccccatca aggcagagca ctagggtgag 116400 tgccaggcag cattccctct ccaggcctac ccatcccagc caggagcagg ctctagatcc 116460 tggtttgttt ccccacccat aataggaagg tgacataata ggacctacgt gggcatcaag 116520 taagttgggg cctgaccacc acaagtgctc attaagtgcc accagctgtt gtgggggtga 116580 tgacactgtg cctccagttc cactcagtct gtgtacttta ttatgccaca gacacatcct 116640 gtactttcct acctcccctt tggctgtgat cccctctact caaaacaaac actcttccct 116700 atcttcattg cattttgttg aaatcccatg gctcttcata gctctcctca gatgcaggcc 116760 cacccccacc cgtgctgttt cctccttgtc tcatcctgcc tgtcacgttc tcctgctcgg 116820 cgggctccac ctcttctgct gccctctagg agatggccag cctttcctgt gctgccactg 116880 ttgtctcacc ttacagtctt cctggctcca gatgagtttg agagcttttg cttatctttg 116940 taacccattt agtatctaac gtggcatttt atacatagga agcttctctc atcagtattg 117000 gtggatgtga accaaattga atactggcag gttggtgaca cggagagcta tgtgcatatg 117060 caaaagctgt agcccctcac ctctggttag ttggccatag gatggagtgt acttaaggta 117120 catagactat tttactccca agaatgctag gcactcactg tcttaattga ggccaccaga 117180 tacacacatg agaatataaa taacggcttg tggcaataat gactaaatgc caaggagtgg 117240 ctggtaaacc gcggtgttcc ctagagaccc cggcctgggc tctacttagg ctgcctcttg 117300 gacatcagac caaggcttac attctgaatc cacagggcat ccacatgggt ggtgtcagtc 117360 ccccacagac agagaagtgt cccgttgcat ttttccatct attccagtag taagattgtg 117420 tcatttgaga ttttctttaa ctgtataatt ggacgtttaa ttaacaaacc agagaggagg 117480 aaaaacaatg aggtgggtag agcatcatgt tcagcctcag ggctgtacag caaagcaatt 117540 ttagactgcg gatgttgagt ctccagttac cctgagtgcc agttacagtg attcacatct 117600 gaaagaacag tactgcagga gagggacagc ccagggtgga tgggtggggt gggcaggagc 117660 tggctggcaa ctccttccct gagctgggcc tgcagagccc tgaggagtgg ggcatgctgt 117720 cctttttgcc tgatttccaa ggattctgct taacgaatta cttcgttcat tttagtaagc 117780 acaggtggct ggtgaagatt ttccagctag gtagatcttt ttgtgtgtgg cttatgactt 117840 ttagggggtg agggaagaaa atagacgaaa atagacttag ttacaaatgt gagtctgtgc 117900 aggaaaatgt ggaggtcagt cgttagttgt gttgtatcaa agacgtgaat gaggaactag 117960 ctgaagtgta agaggttgat tttcctgtac gattaaaaat aaacctgcct ctatgcattt 118020 cagtcgcaat gtatctgctg agcaaaaaga tgaaaacaaa gaagcaaagc ctcgatccct 118080 acgcttcacc tggagcatga aaaccactag ttcaatggat cccggggaca tgatgcggga 118140 aatccgcaaa gtgttggacg ccaataactg cgactatgag cagagggagc gcttcttgct 118200 cttctgcgtc cacggagatg ggcacgcgga gaacctcgtg cagtgggaaa tggaagtgtg 118260 caagctgcca agactgtctc tgaacggggt ccggtttaag cggatatcgg ggacatccat 118320 agccttcaaa aatattgctt ccaaaattgc caatgagcta aagctgtaac ccagtgatta 118380 tgatgtaaat taagtagcaa ttaaagtgtt ttcctgaaca ctgatggaaa tgtatagaat 118440 aatatttagg caataacgtc tgcatcttct aaatcatgaa attaaagtct gaggacgaga 118500 gcacgcctgg gagcgaaagc tggccttttt tctacgaatg cactacatta aagatgtgca 118560 acctatgcgc cccctgccct acttccgtta ccctgagagt cggtgtgtgg ccccatctcc 118620 atgtgcctcc cgtctgggtg ggtgtgagag tggacggtat gtgtgtgaag tggtgtatat 118680 ggaagcatct ccctacactg gcagccagtc attactagta cctctgcggg agatcatccg 118740 gtgctaaaac attacagttg ccaaggagga aaatactgaa tgactgctaa gaattaacct 118800 taagaccagt tcatagttaa tacaggttta cagttcatgc ctgtggtttt gtgtttgttg 118860 ttttgtgttt ttttagtgca aaaggtttaa atttatagtt gtgaacattg cttgtgtgtg 118920 tttttctaag tagattcaca agataattaa aaattcactt tttctcagta aaatcttgca 118980 ttgtctccta atgctgtatt taacacatcc tcaagttgag cagaagtaat gtgtatgaag 119040 gcttggggcc acgtggacct tcgcaggggt cccagcattt tgtgattacc agcatatttt 119100 ttctcccaag gcaataaaga gagaagatgg ccctcatggt gacccaagaa ggaagggaat 119160 ccagagcagc tgcagcttaa ccctgggagt tgtggcacag ccctatggag aaacaccagg 119220 tcttgacagt tctgggttgg ttctctgtga agtatgtaaa tttcctttct cctttcttgt 119280 gattcagtac atgaatcaga cctgcagctt ttgtccaaca cctacatggt tgtgtagggg 119340 taatgagctc ataactcatt gtgttgcttt attctcacag ggaagttgtc acactgattg 119400 gtgtgctatt ctgggttctg ggtttgttgt tggtcagagt atgaaagtct ggaggccgga 119460 cacagtggct cacacctgtg aacccagcac ttttgggagg c 119501 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16 gtctcgttca ttcaccgttg 20 17 20 DNA Artificial Sequence Antisense Oligonucleotide 17 gcgttcagga atatccgcct 20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18 gactggcagt gcctcttggg 20 19 20 DNA Artificial Sequence Antisense Oligonucleotide 19 gagattagtg gagcctcggc 20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 cttgaggtct cgatgtacga 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21 cctcggcttc gagtctggga 20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 ctgcacagga ggctatagag 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 ttaggattac ttgcattccc 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24 gactcctttt cggatacgcc 20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 ttaattctgc acaatgcgag 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 tgtaaggata tgtcttgcca 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 ttctgcacaa tgcgagggtc 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28 ttgaagcaac tggagttctc 20 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29 atctgcacag gaggctatag 20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 gaaaacactt tacttgctac 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 aatggtgtgg cttcatggga 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 gtgtcgagtt taccgccaac 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 tcgtgtcatt ccaccagatg 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34 ctggctggag caattccctt 20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 ttttggtctg agatgtctag 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 tagttctctc actgcaaaca 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 ttttgctcag cagatacatt 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 cacaggaggc tatagagttt 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39 ccgttctaga tcccgggcct 20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 aaagtgctac gactggcagt 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 gtgcaaccaa atagtcaaat 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 cagtgttcag gaaaacactt 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43 cccttgccga ttgttttcaa 20 44 20 DNA Artificial Sequence Antisense Oligonucleotide 44 cttcttcatg ccctgcattg 20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 cgagctcctg agcggctggt 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 ttggatttag caccaggaaa 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 ctgcactact gatactgtgt 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48 agtatttcgt cgtgtcattc 20 49 20 DNA Artificial Sequence Antisense Oligonucleotide 49 gtgtataaaa tgacccccag 20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 agtggagctt ttcttgcgtt 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 cattgcgact ccttgtgagt 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 tagacttgtt ggattcaact 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53 ctagtggaca ttttactgca 20 54 20 DNA Artificial Sequence Antisense Oligonucleotide 54 ttattgcaac ctctctgcct 20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 ctctgaagag cttttgtaga 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 tagcatccag ctctgaagat 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 ctgtgttact actagggaca 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58 atcactgggt tacagcttta 20 59 20 DNA Artificial Sequence Antisense Oligonucleotide 59 tcggcctaac ctctgaagat 20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 ggttgcacat ctttaatgta 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 ggtactagta atgactggct 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 gatgatctcc cgcagaggta 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63 gatgccctta gatgtccggg 20 64 20 DNA Artificial Sequence Antisense Oligonucleotide 64 ccacccgaga ttgagcaata 20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 gatagctctt tctcgattcc 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 ggaaaccaaa gtctttgggt 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 cttgacaacc ctgtctaaat 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68 ctgcagacac aataaatgta 20 69 20 DNA Artificial Sequence Antisense Oligonucleotide 69 gtttagttaa ccaaacacga 20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 cacctttagg tttagttaac 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 ctctcagttc ctttggagag 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 acatgattac ctctagagtg 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73 ccagtatggc atacaaatca 20 74 20 DNA Artificial Sequence Antisense Oligonucleotide 74 tctgataacc gtaatattta 20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 tgacatgttt ctccttgtga 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 agttggaagc cttttgataa 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 ctcatattca gttggaagcc 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78 tgcgacttga gccctcatat 20 79 20 DNA Artificial Sequence Antisense Oligonucleotide 79 tacattgcga cttgagccct 20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 attgtgttac agcagcaaaa 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 gacacatttt tgtgcacctg 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 aatacttcac ctataggtga 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83 gagaagttaa atgatagcca 20 84 20 DNA Artificial Sequence Antisense Oligonucleotide 84 cagacacaat ctgaatagga 20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 tcggcctaac cttagcaagt 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 atcacaatgg tctacatata 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 aggaatagtg ctatgagatc 20 88 20 DNA H. sapiens 88 cccaagaggc actgccagtc 20 89 20 DNA H. sapiens 89 gccgaggctc cactaatctc 20 90 20 DNA H. sapiens 90 tcgtacatcg agacctcaag 20 91 20 DNA H. sapiens 91 tcccagactc gaagccgagg 20 92 20 DNA H. sapiens 92 ctctatagcc tcctgtgcag 20 93 20 DNA H. sapiens 93 gggaatgcaa gtaatcctaa 20 94 20 DNA H. sapiens 94 ggcgtatccg aaaaggagtc 20 95 20 DNA H. sapiens 95 ctcgcattgt gcagaattaa 20 96 20 DNA H. sapiens 96 gaccctcgca ttgtgcagaa 20 97 20 DNA H. sapiens 97 ctatagcctc ctgtgcagat 20 98 20 DNA H. sapiens 98 tcccatgaag ccacaccatt 20 99 20 DNA H. sapiens 99 gttggcggta aactcgacac 20 100 20 DNA H. sapiens 100 catctggtgg aatgacacga 20 101 20 DNA H. sapiens 101 ctagacatct cagaccaaaa 20 102 20 DNA H. sapiens 102 aaactctata gcctcctgtg 20 103 20 DNA H. sapiens 103 aggcccggga tctagaacgg 20 104 20 DNA H. sapiens 104 atttgactat ttggttgcac 20 105 20 DNA H. sapiens 105 ttgaaaacaa tcggcaaggg 20 106 20 DNA H. sapiens 106 caatgcaggg catgaagaag 20 107 20 DNA H. sapiens 107 accagccgct caggagctcg 20 108 20 DNA H. sapiens 108 tttcctggtg ctaaatccaa 20 109 20 DNA H. sapiens 109 acacagtatc agtagtgcag 20 110 20 DNA H. sapiens 110 gaatgacacg acgaaatact 20 111 20 DNA H. sapiens 111 ctgggggtca ttttatacac 20 112 20 DNA H. sapiens 112 aacgcaagaa aagctccact 20 113 20 DNA H. sapiens 113 actcacaagg agtcgcaatg 20 114 20 DNA H. sapiens 114 tgcagtaaaa tgtccactag 20 115 20 DNA H. sapiens 115 tgtccctagt agtaacacag 20 116 20 DNA H. sapiens 116 taaagctgta acccagtgat 20 117 20 DNA H. sapiens 117 tgatttgtat gccatactgg 20 118 20 DNA H. sapiens 118 ggcttccaac tgaatatgag 20 119 20 DNA H. sapiens 119 ttttgctgct gtaacacaat 20 120 20 DNA H. sapiens 120 caggtgcaca aaaatgtgtc 20 121 20 DNA H. sapiens 121 acttgctaag gttaggccga 20 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding MARK3, wherein said compound specifically hybridizes with said nucleic acid molecule encoding MARK3 and inhibits the expression of MARK3.
 2. The compound of claim 1 which is an antisense oligonucleotide.
 3. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified internucleoside linkage.
 4. The compound of claim 3 wherein the modified internucleoside linkage is a phosphorothioate linkage.
 5. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified sugar moiety.
 6. The compound of claim 5 wherein the modified sugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. The compound of claim 2 wherein the antisense oligonucleotide comprises at least one modified nucleobase.
 8. The compound of claim 7 wherein the modified nucleobase is a 5-methylcytosine.
 9. The compound of claim 2 wherein the antisense oligonucleotide is a chimeric oligonucleotide.
 10. A compound 8 to 80 nucleobases in length which specifically hybridizes with at least an 8-nucleobase portion of a preferred target region on a nucleic acid molecule encoding MARK3.
 11. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.
 12. The composition of claim 11 further comprising a colloidal dispersion system.
 13. The composition of claim 11 wherein the compound is an antisense oligonucleotide.
 14. A method of inhibiting the expression of MARK3 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of MARK3 is inhibited.
 15. A method of treating an animal having a disease or condition associated with MARK3 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of MARK3 is inhibited.
 16. The method of claim 15-wherein the disease or condition is a hyperproliferative disorder.
 17. The method of claim 16 wherein the hyperproliferative disorder is cancer.
 18. The method of claim 15 wherein the disease or condition is a neurodegenerative disorder.
 19. The method of claim 18 wherein the neurodegenerative disorder is Alzheimer's disease.
 20. A method of screening for an antisense compound, the method comprising the steps of: a. contacting a preferred target region of a nucleic acid molecule encoding MARK3 with one or more candidate antisense compounds, said candidate antisense compounds comprising at least an 8-nucleobase portion which is complementary to said preferred target region, and b. selecting for one or more candidate antisense compounds which inhibit the expression of a nucleic acid molecule encoding MARK3. 